Bioconjugates and delivery of bioactive agents

ABSTRACT

The present invention relates to bioconjugates and the delivery of bioactive agents which are preferably targeted for site-specific release in cells, tissues or organs. More particularly, this invention relates to bioconjugates which comprise a bioactive agent and an organocobalt complex. The bioactive agent is covalently bonded directly or indirectly to the cobalt atom of the organocobalt complex. The bioactive agent is released from the bioconjugate by the cleavage of the covalent bond between the bioactive agent and the cobalt atom in the organocobalt complex. The cleavage may occur as a result of normal displacement by cellular nucleophiles or enzymatic action, but is preferably caused to occur selectively as a predetermined release site by application of an external signal. The external signal may be light or photoexcitation, i.e. photolysis, or it may be ultrasound, i.e. sonolysis. Further, if the photolysis takes place in the presence of a magnetic field surrounding the release site, the release of the bioactive agent into surrounding healthy tissue is minimized.

[0001] This invention was made in part with Government support underGrant No. ES05728 awarded by the National Institutes of Health,Bethesda, Md. The United States Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to bioconjugates and the deliveryof bioactive agents which are preferably targeted for site-specificrelease in cells, tissues or organs. More particularly, this inventionrelates to bioconjugates which comprise a bioactive agent and anorganocobalt complex. The bioactive agent is covalently bonded directlyor indirectly to the cobalt atom of the organocobalt complex. Thebioactive agent is released from the bioconjugate by the cleavage of thecovalent bond between the bioactive agent and the cobalt atom in theorganocobalt complex. The cleavage may occur as a result of normaldisplacement by cellular nucleophiles or enzymatic action, but ispreferably caused to occur selectively at a predetermined release siteby application of an external signal. The external signal may be lightor photoexcitation, i.e. photolysis, or it may be ultrasound, i.e.sonolysis. Further, if the photolysis takes place in the presence of amagnetic field surrounding the release site, the release of thebioactive agent into surrounding healthy tissue is minimized.

[0003] The publications and other materials used herein to illuminatethe background of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedbibliography.

[0004] The focus of a substantial body of research has been thedevelopment of a system whereby a pharmaceutical agent can beselectively delivered to a desired anatomic location; namely the site inneed of treatment. In spite of the great progress which has beenachieved in this regard, many pharmaceutical delivery systems for thetreatment of various diseases or health risks, e.g., the treatment ofcancer, impart substantial risk to the patient. With respect to thetreatment of cancer, drugs which are effective in attacking malignantcells to destroy them, or at least limit their proliferation, have atendency to attack benign cells also. Therefore, it is highly desirableto limit the location of their action to that of the malignancy, and toensure that at any particular time effective, but not excessive, amountsof such drugs are used.

[0005] Although it is desired to concentrate a cytotoxic agent at atargeted site, current cancer treatment protocols for administeringthese cytotoxic agents typically call for repeated intravenous dosing,with careful monitoring of the patient. The drugs are often used incombination to exert a multi-faceted assault on neoplastic cells. Thedose is selected to be just below the amount that will produce acute(and sometimes chronic) toxicity that can lead to life-threateningcardiomyopathy, myelotoxicity, hepatic toxicity, or renal toxicity.Alopecia (hair loss), mucositis, stomatitis, and nausea are othercommon, but generally not life-threatening, side effects at these doses.Since many of these compounds are potent vesicants, tissue necrosis willoccur if localized extravasation (loss of the drug from blood to thesurrounding tissue) occurs. These effects occur since the bloodgenerally attains a specified concentration of that drug before becomingeffective. Because the blood is transported throughout the body of thehost being treated, so is the pharmaceutical agent. Following thistechnique provides an even distribution of the drug throughout the body,rather than concentrating it at the treatment site. Moreover, suchsystemic treatment methods expose the healthy cells to the cytotoxicagent concurrent with the treatment of the unhealthy or diseased cellsbesides limiting the concentration of the drug at the site where it ismost needed.

[0006] Previous attempts to administer such drugs by direct injectioninto the location of the organ having the malignancy are only partiallyeffective, because of migration of the drug from that location and as aresult of extensive tissue necrosis from extravasation. Such dispersioncannot be totally prevented, with the result that excessive quantitiesof drug need to be administered to attain a desired result. Althoughcareful clinical monitoring may prevent extensive damage or loss ofviable tissue, the providing of a pharmaceutical agent-carrier systemwhich is actively transported through standard biological systems to thetreatment site prior to activation of the pharmaceutical agent would behighly desirable not only in optimizing utilization of the drug but alsoin the reduction of side effects and/or the minimization of thedestruction of healthy cells. The direct injection of cytotoxic agentsinto solid tumors of the breast, bladder, prostate and lung usingconventional cytotoxic chemotherapeutic agents as adjuvants to surgeryand/or radiotherapy has been of limited success in prolonging the livesof patients. This is partially due to the dose limitations imposed bythe acute and chronic toxicity to tissues or organ systems beyond thosethat are targeted.

[0007] As it relates to the administration of cytotoxic orantineoplastic drugs, the effective resolution of concerns relating tomodes of administration, to the limitation of dosage size and frequencyof administration, and to side effects would certainly be of benefit tothe treatment of cancer.

[0008] Oligonucleotides that specifically interfere with gene expressionat the transcriptional or translational levels have the potential to beused as therapeutic agents to control the synthesis of deleteriousproteins associated with viral, neoplastic or other diseases. It ispossible to select single-stranded oligonucleotides that recognize andbind to the major groove of a stretch of double-stranded DNA in asequence-specific manner to form a triple helix (Le Doan et al., 1987;Moser and Dervan, 1987). Triple helix-forming oligonucleotides targetedto the promoter region of certain genes have been used to physicallyblock RNA synthesis in cell-free transcription assays (Cooney et al.,1988; Postel et al., 1992; Skoog et al., 1993; Rando et al., 1994).Similarly, in vitro translation assays have been used to demonstratethat antisense oligonucleotides can bind mRNA targets and preventprotein synthesis (Uhlmann and Peyman, 1990; Cohen and Hogan, 1994).

[0009] Although antisense oligonucleotides have shown great efficacy inthe selective inhibition of gene expression (Stein and Cohen, 1988;Szczylik et al., 1991; Gray et al., 1993), the therapeutic applicationsof such antisense oligonucleotides are currently limited by their lowphysiological stability, slow cellular uptake, and lack of tissuespecificity. The instability obstacles have been largely overcome by useof backbone-modified oligonucleotides that are more resistant tonucleases. Methylphosphonates, protein-nucleic acid conjugates, andphosphorothioates all appear to resist enzymatic digestion better thanthe corresponding natural oligonucleotides (Chang and Miller, 1991;Wickstrom et al., 1992; Letsinger, 1993; Zon, 1993).

[0010] Problems with cellular uptake of antisense oligonucleotides havebeen more difficult to solve. Endogenous uptake pathways that rely onpinocytosis and related processes generally have insufficient capacityto deliver the quantities of antisense of oligonucleotides required tosuppress gene expression (Vlassov et al., 1994). Hydrophobicmodifications have also been undertaken to improve membranepermeability, but such derivatization strategies are most useful onlyfor short olgonucleotides (Vlassov et al., 1994). Although complexes ofantisense constructs with cationic liposomes or immunoliposomes (Gao andHuang, 1991; Bennett et al., 1992, Ma and Wei, 1996) and polylsine(Trubetskoy et al., 1992; Bunnell et al., 1992) have significantlyenhanced intracellular delivery, they have simultaneously introduced newdisadvantages of their own. Thus, both methods exhibit some carriercytotoxicity, and like other protocols, neither strategy allows for anytissue or cell targeting. In short, intracellular delivery and tissuespecificity remain major obstacles to the implementaiton of antisensedrugs in the treatment of human disorders.

[0011] Other techniques for the delivery of oligonucleotides to cellsinclude the use of: (a) folate-PEG-liposome constructs for the deliveryof antisense DNA against growth factor receptor (Wang et al., 1995); (b)folic acid-polylysine constructs for the delivery of c-myc antisense DNA(Ginobbi et al., (1997); (c) tris(N-acetylgalactosamine aminohexylglycoside) amide of tyrosyl(glutamyl)-glutamate (YEE(GaINAcAH)₃) linkedto polylysine for the delivery of DNA to cells via theasialoglycoprotein receptor (Merwin et al., 1994); and (d) water-solubleblock polycations (Kabanov et al., 1995).

[0012] It has been known for some time that a pharmaceutically activeagent can be attached to a carrier or molecule. The term “prodrug” isoften associated with such systems wherein the active agent is bonded toanother molecule for purposes of administration. The drug is usuallyinactive in the prodrug state and the bond is later cleaved releasingthe drug at a site where it can be effective. However, such systems arenot as useful as might be desired for various reasons, site specificitybeing one. Also, the release of the drug from its carrier requires thepresence of some agent or event to separate the active drug from itscarrier or molecule and, as such, may rely on factors such as thepresence of a specific enzyme, pH conditions, time release and the like,which may be variable from host to host and which may not be effectivelyimplemented.

[0013] For example, transmembrane transport of nutrient molecules is acritical cellular function. Because practitioners have recognized theimportance of transmembrane transport to many areas of medical andbiological science, including drug therapy, peptide therapy and genetransfer, there have been significant research efforts directed to theunderstanding and application of such processes. Thus, for example,transmembrane delivery of nucleic acids has been encouraged through theuse of protein carriers, antibody carriers, liposomal delivery systems,electroporation, direct injection, cell fusion, vital carriers, osmoticsh ck, and calcium-phosphate mediated transformation. However, many ofthose techniques are limited both by the types of cells in whichtransmembrane transport is enabled and by the conditions of use forsuccessful transmembrane transport of exogenous molecular species.Further, many of these known techniques are limited in the type and sizeof exogenous molecule that can be transported across a membrane withoutloss of bioactivity.

[0014] One method for transmembrane delivery of exogenous moleculeshaving a wide applicability is based on the mechanism ofreceptor-mediated endocytotic activity. Unlike many other methods,receptor-mediated endocytotic activity can be used successfully both invivo and in vitro. Receptor-mediated endocytosis involves the movementof ligands bound to membrane receptors into the interior of an areabounded by the membrane through invagination of the membrane. Theprocess is initiated or activated by the binding of a receptor-specificligand to the receptor. Many receptor-mediated endocytotic systems havebeen characterized, including those recognizing galactose, mannose,mannose 6-phosphate, transferrin, asialoglycoprotein, transcobalamin(Vitamin B₁₂), α-2-macroglobulins, insulin, and other peptide growthfactors such as epidermal growth factor (EGF).

[0015] Receptor-mediated endocytotic activity has been utilized fordelivering exogenous molecules such as proteins and nucleic acids tocells. Generally, a specified ligand is chemically conjugated bycovalent, ionic or hydrogen bonding to an exogenous molecule of interest(i.e. the exogenous compound), forming a conjugate molecule having amoiety (the ligand portion) that is still recognized in the conjugate bya target receptor. Using this technique, the phototoxic agent psoralenhas been conjugated to insulin and internalized by the insulin receptorendocytotic pathway (Gasparro, 1986); the hepatocyte-specific receptorfor galactose terminal asialoglycoproteins has been utilized for thehepatocyte-specific transmembrane delivery ofasialoorosomucoid-poly-L-lysine non-covalently complexed to a DNAplasmid (Wu, 1987); the cell receptor for epidermal growth factor hasbeen utilized to deliver polynucleotides covalently linked to EGF to thecell interior (Myers, 1988); the intestinally situated cellular receptorfor the organometallic Vitamin B₁₂-intrinsic factor complex has beenused to mediate delivery to the circulatory system of a vertebrate hosta drug, hormone, bioactive peptide or immunogen complexed with VitaminB₁₂ and delivered to the intestine through oral administration(Russell-Jones et al., 1995); the mannose-6-phosphate receptor has beenused to deliver low density lipoproteins to cells (Murray and Neville,1980); the cholera toxin binding subunit receptor has been used todeliver insulin to cells lacking insulin receptors (Roth and Maddox,1983); the human chorionic gonadotropin receptor has been employed todeliver a ricin a-chain coupled to HCG to cells with the appropriate HCGreceptor in order to kill the cells (Oeltmann and Heath, 1979); thetransferrin receptor has been used to deliver mitomycin C to sarcomacells (Tanaka et al., 1996) or to deliver doxorubicin tomultidrug-resistant cells (Fritzer et al., 1996);the biotin receptor hasbeen employed to deliver hypoxanthine-guanine phosphoribosyl transferase(HGPRT) by biotinylating the HGPRT to restore growth to HGPRT deficientcells (Low et al., 1995); and the folic acid receptor has been used todeliver antisense DNA to src-transformed fibroblast cells (Low et al.,1995).

[0016] Russell-Jones et al. (1995), describes a system which involvesthe formation of a covalent bond between the pharmaceutical agent onewishes to deliver and a modified Vitamin B₁₂ to form a conjugatemolecule. The conjugate is orally administered and is then transportedfrom the intestinal lumen to the circulation. Importantly, thepharmaceutical agent and the vitamin are bound through an amide linkagewhich is prone to acid hydrolysis. Russell-Jones et al. found that manybiologically active pharmaceutical agents can be bound to B₁₂ forfacilitating the introduction of the drug into the blood stream throughoral administration. Importantly, no method was provided whereby thedrug-B₁₂ bond could be selectively cleaved, nor could location of theactive pharmaceutical agent be controlled once activated. Instead,Russell-Jones et al. relied on biochemical degradation of the drug-B₁₂bond to release the drug in its active form. Importantly, under thismethod the drug could be released in its active form anywhere within thecirculation system, diminishing the importance of the active transportof B₁₂ into cancer tissue. Moreover, the conjugates formed under thismethod require the modification of the structure of the corrin ring ofthe B₁₂ molecule, which modification can have serious effects onreceptor interactions.

[0017] Thus, there exists a need for a drug delivery system which can beutilized for the delivery of bioactive agents, includingpharmaceuticals, peptides and oligonucleotides. There is also a need fora drug delivery system which can be used for site-specific release ofthe bioactive agent in the cells, tissues, or organs in which atherapeutical effect is desired to be effected.

SUMMARY OF THE INVENTION

[0018] The present invention relates to bioconjugates and the deliveryof bioactive agents which are preferably targeted for site specificrelease in cells, tissues or organs. More particularly, this inventionrelates to bioconjugates which comprise a bioactive agent and anorganocobalt complex. The bioactive agent is covalently bonded directlyor indirectly to the cobalt atom of the organocobalt complex. Thebioactive agent is released from the bioconjugate by the cleavage of thecovalent bond between the bioactive agent and the cobalt atom in theorganocobalt complex, as described herein.

[0019] The bioactive agent is any agent which is desired to be deliveredto cells, tissues or organs for nutrient or therapeutic effects. Inaccordance with the present invention, bioactive agents include, but arenot limited to, nutrients, pharmaceuticals, drugs, peptides andoligonucleotides.

[0020] The organocobalt complex is any organic complex containing acobalt atom having bound thereto 4-5 nitrogen and/or chalcogens such asoxygen, sulfur, etc., as part of a multiple unsaturated heterocyclicring system. In accordance with the present invention, suitableorganocobalt complexes include, but are not limited to, cobalamin,Co[SALEN], organo-(pyridine)bis(dimethylglyoximato)cobalt, corrinoids,derivatives thereof and analogues thereof.

[0021] The organocobalt complexes may be unsubstituted or substitutedwith conventional organic functional groups which will not alter thebasic nature of the organocobalt complex. The basic is nature of theorganocobalt complex is to directly or indirectly bind the bioactiveagent covalently to the cobalt such that the cobalt-bioactive agent bondis readily cleavable as described herein. The organocobalt complex mayalso be covalently bound directly or indirectly to a targeting molecule.The targeting molecule is a molecule for which the desired cell, tissueor organ has a requirement or a receptor, as described herein.

[0022] The bioconjugate according to the present invention isadministered to a subject in need of therapeutic treatment. Thebioconjugate concentrates in a targeted cell, tissue or organ site as aresult of the organocobalt complex. As an example, a bioconjugatecontaining a chemotherapeutic is administered to a patient and thebioconjugate concentrates in neoplastic cells where the activechemotherapeutic is released from the bioconjugate by cleavage.Similarly, other pharmaceuticals, drugs, peptides or oligonucleotidesare administered to a subject as part of the bioconjugate which isconcentrated in the desired cells, tissues or organs. Thepharmaceuticals, drugs, peptides or oligonucleotides are released bycleavage. In one embodiment, the cleavage may occur as a result ofnormal displacement by cellular nucleophiles or enzymatic aciton. In asecond embodiment, the cleavage is caused to occur selectively at therelease site by an external signal. The external signal may be light orphotoexcitation, i.e. photolysis, or it may be ultrasound, i.e.sonolysis. Further, if the photolysis takes place in the presence of amagnetic field surrounding the release site the release of the drug,such as a cytotoxic agent, into surrounding healthy tissue can beminimized.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 shows the structure and absorption spectrum ofmethylcobalamin (B₁₂).

[0024]FIG. 2 shows the structure and absorption spectrum ofethyl-Co[SALEN] (cobalt-bis-[salicylidene]-ethylenediamine.

[0025]FIG. 3A shows a sequential absorption spectra of aqueousCH₃-Cbl^(III) as a function of anaerobic sonolysis (pH 7.38, 100 mMHepes, saturating Ar).

[0026]FIG. 3B shows the change in absorbance spectra following aerobicsonolysis in the absence of organic buffer.

[0027]FIG. 4A shows a sequential absorption spectra of aqueous compound3 (Example 6) as a function of anaerobic sonolysis at pH 7.4, 100 mMHepes, saturating Ar.

[0028]FIG. 4B shows the change in absorbance spectra following aerobicsonolysis of a compound 3 (Example 6) solution containing phosphatebuffer.

[0029]FIG. 5 shows the effect of a chlorambucil bioconjugate on cellviability for the HCT-116 cell line. The results are shown forchlorambucil (▪), the chlorambucil bioconjugate with photolysis (∘), thechlorambucil bioconjugate with no photolysis (▴) and the chlorambucilbioconjugate plus 10 equivalents of hydroxycobalamin with photolysis(∇).

[0030]FIG. 6 shows the effect of a chlorambucil bioconjugate on cellviability for the HL-60 cell line. The results are shown forchlorambucil (▪), the chlorambucil bioconjugate with no photolysis (▴)and the chlorambucil bioconjugate plus 10 equivalents ofhydroxycobalamin with no photolysis (∇).

[0031]FIG. 7 shows the effect of a chlorambucil bioconjugate on cellviability for the B-16 cell line. The results are shown for chlorambucil(▪), the chlorambucil bioconjugate with photolysis (∘), the chlorambucilbioconjugate with no photolysis (▴) and the chlorambucil bioconjugateplus 10 equivalents of hydroxycobalamin with photolysis (∇).

[0032]FIG. 8 shows the effect of a chlorambucil bioconjugate on cellviability for the Meth-A cell line. The results are shown forchlorambucil (▪), the chlorambucil bioconjugate with photolysis (∘), thechlorambucil bioconjugate with no photolysis (▴) and the chlorambucilbioconjugate plus 10 equivalents of hydroxycobalamin with photolysis(∇).

[0033]FIG. 9 shows the effect of a chlorambucil bioconjugate on cellviability for the RD-995 cell line. The results are shown forchlorambucil (▪), the chlorambucil bioconjugate with photolysis (∘), thechlorambucil bioconjugate with no photolysis (▴) and the chlorambucilbioconjugate plus 10 equivalents of hydroxycobalamin with photolysis(∇).

DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention relates to bioconjugates and the deliveryof bioactive agents which are preferably targeted for site-specificrelease in cells, tissues or organs. More particularly, this inventionrelates to bioconjugates which comprise a bioactive agent and anorganocobalt complex. The bioactive agent is covalently bonded directlyor indirectly to the cobalt atom of the organocobalt complex. Thebioactive agent is released from the bioconjugate by the cleavage of thecovalent bond between the bioactive agent and the cobalt atom in theorganocobalt complex. The cleavage may occur as a result of normaldisplacement by cellular nucleophiles or enzymatic action, but ispreferably caused to occur selectively at a predetermined release siteby application of an external signal. The external signal may be lightor photoexcitation, i.e. photolysis, or it may be ultrasound, i.e.sonolysis. Further, if the photolysis takes place in the presence of amagnetic field surrounding the release site, the release of thebioactive agent into surrounding healthy tissue is minimized.

[0035] The bioconjugate according to the present invention isadministered to a subject in need of therapeutic treatment. Thebioconjugate concentrates in a targeted cell, tissue or organ site as aresult of the organocobalt complex. The bioactive agent is released fromthe bioconjugate by cleavage. In one embodiment, the cleavage may occuras a result of normal displacement by cellular nucleophiles or enzymaticaciton. In a second embodiment, the cleavage is caused to occurselectively at the release site by an external signal. The externalsignal may be light or photoexcitation, i.e. photolysis, or it may beultrasound, i.e. sonolysis. Further, if the photolysis takes place inthe presence of a magnetic field surrounding the release site, therelease of the drug, such as a cytotoxic agent, into surrounding healthytissue is minimized.

[0036] As one example, the bioconjugate contains a chemotherapeuticagent and is administered to a patient having cancer. In this example, atherapeutically effective amount of the bioconjugate is administeredintravenously to a patient such that the bioconjugate concentrates inthe neoplastic cells. The chemotherapeutic agent is released from thebioconjugate by natural means (e.g., cellular nucleophiles or enzymaticaction) or preferably by means of an external signal (e.g., light orultrasound).

[0037] As a second example, the bioconjugate contains a cytotoxic agentand is administered to a patient having psoriasis. In this example, atherapeutically effective amount of the bioconjugate is administered toan afflicted skin site. The cytotoxic agent is released by natural meansor preferably by means of an external signal.

[0038] As a third example, the bioconjugate contains the enzymaticdomain of diphtheria toxin (Nichols et al., 1997) and is administered toa patient having cancer. In this example, a therapeutically effectiveamount of the bioconjugate is administered intravenously to a patientsuch that the bioconjugate concentrates in the neoplastic cells. Theenzymatic domain of diphtheria toxin is released from the bioconjugateby natural means (e.g., cellular nucleophiles or enzymatic action) orpreferably by means of an external signal (e.g., light or ultrasound)and proceeds to kill the cancer cells.

[0039] As a fourth example, the bioconjugate contains an antisenseoligonucleotide against hepatitis B virus (Yao et al., 1996; Madon andBlum, 1996) and is administered to a subject having hepatitis B. In thisexample, a therapeutically effective amount of the bioconjugate isadministered intravenously to a patient such that the bioconjugateconcentrates in the liver. The antisense oligonucleotide is releasedfrom the bioconjugate by natural means (e.g., cellular nucleophiles orenzymatic action) or preferably by means of an external signal (e.g.,light or ultrasound) and proceeds to inhibit gene expression andreplication of hepatitis B virus.

[0040] The present invention employs the following definitions:

[0041] Bioactive agent: any agent which is desired to be delivered tocells, tissues or organs for modulating or otherwise modifying cellfunction, including for therapeutic effects. In accordance with thepresent invention, bioactive agents include, but are not limited to,pharmaceutically active compounds or diagnostic compounds. Bioactiveagents include, but are not limited to, peptides, oligopeptides,proteins, apoproteins, glycoproteins, antigens and antibodies orantibody fragments thereto, receptors anti other membrane proteins,retro-inverso oligopeptides, protein analogs in which at least onenon-peptide linkage replaces a peptide linkage, enzymes, coenzymes,enzyme inhibitors, amino acids and their derivatives, hormones, lipids,phospholipids, liposomes, ricin or ricin fragments; toxins such asaflatoxin, digoxin, xanthotoxin, rubratoxin; antibiotics such ascephalosporins, penicillin and erythromycin; analgesics such as aspirin,ibuprofen and acetaminophen, bronchodilators such as theophylline andalbuterol; beta-blockers such as propranolol, metoprolol, atenolol,labetolol, timolol, penbutolol and pindolol; antimicrobial agents suchas those described above and ciprofloxacin, cinoxacin and norfloxacin;antihypertensive agents such as clonidine, methyldopa, prazosin,verapamil, nifedipine, aptopril and enalapril; cardiovascular agentsincluding antiarrhythmics, cardiac glycosides, antianginals andvasodilators, central nervous system agents including stimulants,psychotropics, antimanics and depressants; antiviral agents;antihistamines such as chlorphenirmine and brompheniramine; cancer drugsincluding chemotherapeutic agents, such as chlorambucil, carboplatin,deratives of busulfan, doxorubicin, etoposide, topotecan (TPT);tranquilizers such as diazepam, chordiazepoxide, oxazepam, alprazolamand triazolam, anti-depressants such as fluoxetine, amitriptyline,nortriptyline and imipramine; H-2 antagonists such as nizatidine,cimetidine, famotidine and ranitidine, anticonvulsants; antinauseants;prostaglandins; muscle relaxants; anti-inflammatory substances;stimulants; decongestants; antiemetics; diuretics; antispasmodics;antiasthmatiics; anti-Parkinson agents; expectorants; coughsuppressants, mucolytics; vitamins; and mineral and nutritionaladditives. Other molecules include nucleotides; oligonucleotides;polynucleotides; and their art-recognized and biologically functionalanalogs and derivatives including, for example, methylatedpolynucleotides and nucleotide analogs having phosphorothioate linkages;plasmids, cosmids, artificial chromosomes, other nucleic acid vectors;antisense polynucleotides including those substantially complementary toat least one endogenous nucleic acid or those having sequences with asense opposed to at least portions of selected viral or retroviralgenomes; promoters; enhancers; inhibitors; other ligands for regulatinggene transcription and translation. In addition, the bioactive agent canbe any other biologically active molecule that can form a conjugate withan organocobalt complex. The bioactive agent may further contain aspacer which provides a covalent bond with the cobalt atom of theorganocobalt complex, but which does not adversely affect the biologicalactivity of the bioactive agent.

[0042] Bioconjugate: a conjugate containing a bioactive agent and anorganocobalt complex in which the bioactive agent is covalently bounddirectly to the cobalt atom or is covalenly bound indirectly to thecobalt atom via a spacer.

[0043] Non-reactive atom: an atom in the bioactive agent that will notlead to rearrangement or destruction of the bioactive agent underconditions of ligand exchange during receptor-mediated endocytosis, butthat instead will reproduce the original form of the bioactive agent (orbioactive agent and spacer) and thereby unmask an active bioactiveagent. The non-reactive atom may be a carbon atom, a nitrogen atom, anoxygen atom, a sulfur atom, a selenium atom or a silicon atom. A carbonatom (e.g. from an alkyl, acyl or aryl group) is particularly preferred.Such non-reactive atoms are also used in forming the covalent bondbetween the cobalt and the spacer.

[0044] Organocobalt complex: an organic complex containing a cobalt atomhaving bound thereto 4-5 calcogens as part of a multiple unsaturatedheterocyclic ring system. In accordance with the present invention,suitable organocobalt complexes include, but are not limited to,cobalamin (coenzyme B₁₂), Co[SALEN] (which is a cobalamin analogue),organo(pyridine)-bis(dimethylglyoximato)cobalt, corrinoids (such asdisclosed by Brown et al., 1996) and derivatives or analogues of any ofthe preceding, as well as pharmaceutically acceptable salts. Theorganocobalt complexes may be unsubstituted or substituted withconventional organic functional groups which will not alter the basicnature of the organocobalt complex. The basic nature of the organocobaltcomplex is to bind the bioactive agent covalently to the cobalt suchthat the cobalt-bioactive agent bond is readily cleavable as describedherein. Examples of substituents which may be found on the organocobaltcomplex include amino, nitro, halogen (bromine, chlorine), sulfito,C₂₋₆-alkene and C₂₋₆ alkyne. For example, the organocobalt complex canbe formed having a nitro and/or halo (e.g., bromo) derivative of thecorrin ring or having an extended conjugation with exocyclic olefin oralkylene groups. Other derivatives include cobalamin lactone, cobalaminlactame and those in which the benzimidiazole ring (e.g., of cobalamin,green corrinoid, and the like) are substituted with e.g., one or morehalogen (bromine, chlorine), hydroxy or C₁₋₆ alkyl. Such substituentsare useful for increasing the λ_(max) to be used for cleavage of thebioconjugate as described herein. Further derivatives include anilide,ethylamide, mono-, di- or tri-carboxylic acid or proprionamidederivatives of cobalamin of Vitamin B₁₂. In one embodiment, theorganocobalt complex is any organic complex conaining cobalt which isbound by transcobalamin and transported into a cell by areceptor-mediated process involving transcobalamin. In a secondembodiment, the organocobalt complex may also be covalently bounddirectly or indirectly (through a spacer) to a targeting molecule,wherein said targeting molecule is bound by its receptor and the complexis transported into a cell by a receptor-mediated process. Co[SALEN] andits derivatives or analogues can be represented by the general formula

[0045] wherein the substitutents may be included or omitted to modulatephysical properties of the molecule, e.g., water solubility, stabilityor λ_(max)—the wavelength at which the complex absorbs. Thus, thesubstituents are as follows: R is H, a group which increases watersolubility and/or stability or a group for attachment of a targetingmolecule and W, W′, X, X′, Y, Y′, Z and Z′ are independently H, a groupwhich increases water solubility and/or stability, a group forattachment of a targeting molecule or a group for modified absorbance ofenergy, or W and X together and W′ and X′ together are a 4-6 membercyclic or heterocyclic ring, or Y and Z together and Y′ and Z′ togetherare a 4-6 member cyclic or heterocyclic aromatic ring. Examples ofgroups for enhancing water solubility include amino, C₁₋₆ alcohol, C₁₋₆carboxyl for any substitutent, or also SO₃— for the substitutents otherthan R. Examples of groups for attachment of a targeting moleculeinclude amino, C₁₋₆ alcohol and C₁₋₆ carboxyl for any substitutent.Examples of groups for modifying absorbance include CH₂OH, CO₂H, SO₃—,amino and nitro for the substitutents other than R. Such groups areuseful for increasing the wavelength of light to be used for cleavage ofthe bioconjugate as described herein, while targeting molecules areuseful in selectively targeting the bioconjugate to the desired tissue.Therefore, when used in the context of the present application, the termorganocobalt complex, unless specifically identified, shall be inclusiveof B₁₂ in all its embodiments, including coenzyme B₁₂, Co[SALEN] andother B₁₂ or B₁₂-like molecules, the organocobalt complexes definedherein, as well as any derivatives and analogues thereof.

[0046] Spacer: an atom or molecule which covalently binds together twocomponents. In the present invention, a spacer is intended to includeatoms and molecules which can be used to covaltently bind a bioactiveagent to the cobalt atom of an organocobalt complex or to covalentlybind a targeting molecule to an organocobalt complex. The spacer mustnot prevent the binding of the organocobalt complex or the targetingmolecule with its appropriate receptor. Examples of suitable spacersinclude, but are not limited to, polymethylene [—(CH₂)_(n), where n is1-10], ester [bioactive agent attached to O and Co to C═O], carbonate,ether, acetal or any combination of two or more of these units. Askilled artisan will readily recognize other spacers which can be usedin accordance with the present invention.

[0047] Several of these spacers are useful as a “self-destructing”linker group. That is, some or all of the linkage would be consumed in afragmentation reaction. This means that, following cleavage of the C—Cobond by photolysis or sonolysis, an additional cleavage will take placeseveral bonds away, leading to the formation of a small, unsaturated(and typically volatile) molecule made up of atoms of the former linker.This is shown schematically below:

[0048] The most typical scenario is the subsequent cleavage of a secondbond, two bonds removed from the first. Thus, most self-destructivelinkers would contain a two-atom unit whose extrusion as a small,gaseous molecule is favorable. Another design feature is to have the newradical species which is generated after the second cleavage step be anespecially stable kind of radical. Examples of self-destructing linkersare shown below:

[0049] Targeting Molecule: a molecule which is bound by a receptor andtransported into a cell by a receptor-mediated process. Examples ofsuitable targeting molecules include, but are not limited to, glucose,galactose, mannose, mannose 6-phosphate, transferrin,asialoglycoprotein, α-2-macroglobulins, insulin, a peptide growthfactor, cobalamin, folic acid or derivatives, biotin or derivatives,YEE(GalNAcAH)₃ or derivatives, albumin, texaphyrin, metallotexaphyrin,porphyrin, any vitamin, any coenzyme, an antibody, an antibody fragment(e.g., Fab) and a single chain antibody variable region (scFv). Askilled artisan will readily recognize other targeting molecules(ligands) which bind to cell receptors and which are transported into acell by a receptor-mediated process. The present invention is intendedto include all such targeting molecules.

[0050] The present invention takes advantage of the cellular propertiesof cobalamin and cobalamin analogues or derivatives, as well as thecellular properties of other targeting molecules. For example, studieshave shown that the absorption of physiological amounts of vitamin B₁₂by the gut requires that it be complexed with a naturally occurringtransport protein known as intrinsic factor (IF). (Castle, 1953; Fox andCastle, Allen and Majerus, 1972b). This protein is released into thelumen of the stomach by parietal cells in the fundus. Once bound tointrinsic factor, the B₁₂-IF complex interacts with a membrane boundreceptor for IF located on the terminal ileum of the small intestine.The receptor-IF-B₁₂ complex is then internalized by a process ofreceptor-mediated endocytosis (RME). Allen and Majerus demonstrated thatit is possible to chemically modify B₁₂, couple it to a resin and usethe B₁₂-resin to affinity purify IF (Allen and Majerus, 1972a). Thisfinding suggests the possibility of coupling a large macromolecule (suchas the resin used by Allen and Majerus, 1972a) to B₁₂ while stillpreserving its ability to interact specifically with intrinsic factorand thus be part of the active transport system. By coupling moleculesto B₁₂ in such a way as to preserve the ability of B₁₂ to interact withintrinsic factor, it was found that the natural uptake mechanism fororally administered B₁₂ could be used to deliver various proteins, drugsor other pharmaceutically active molecules from the intestinal lumen tothe circulation. It has been found that B₁₂ is naturally concentrated incancer tissue through a similar transport mechanism.

[0051] In mammals, B₁₂ is transported in the blood by transcobalaminproteins TC-I, TC-II, and TC-III. The major form of B₁₂ in the blood ismethylcobalamin and the largest store of B₁₂ is adenosylcobalamin in theliver. Rapidly dividing cells, including cancer cells, require coenzymeB₁₂ for thymidine production during DNA synthesis. It has been reportedby Carmel (1975) that, in some patients with tumors, up to 50-foldincreases in the major cobalamin transport proteins TC-I and TC-II havebeen observed. Waxman et al. (1972), report the finding of tumorspecific B₁₂ binding proteins that circulate in the blood. In eachinstance, these increases in TC transport proteins and the correspondingsystemic aepletion of B₁₂ were not the result of megaloblastosis,granulocyte proliferation, or any other pathogenic B₁₂ deficiency.

[0052] In a second example of receptor-mediated endocytosis, folatereceptors that mediate endocytotic activity have previously beenidentified in bacterial cells (Kumar et al., 1987) and used for deliveryof biologically active materials (Low et al., 1995). Folic acid, folinicacid, pteropolyglutamic acid, and folate receptor-binding pteridinessuch as tetrahydropterins, dihydrofolates, tetrahydrofolates and theirdeaza and dideaza analogs are useful as targeting molecules inaccordance with the present invention. The terms “deaza” and “dideaza”analogs refer to the art-recognized analogs having a carbon atomsubstituted for one or two nitrogen atoms in the naturally-occurringfolic acid structure. For example, the deaza analogs include the1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs. The dideazaanalogs include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza,and 5,8-dideaza analogs. The foregoing folic acid deriatives areconventionally termed “folates,” reflecting their capacity to bind withfolate-receptors, and such ligands when complexed with exogenousmolecules are effective to enhance trans-membrane transport. Otherfolates useful as complex forming ligands for this invention are thefolate receptor binding analogs aminopterin, amethopterin(methotrexate), N-methylfolate, 2-deamino-hydroxyfolate, deaza analogssuch as 1-deazamethopterin or 3-deazamethopterin, and3′5′-dichloro-4-amino-4-deoxy-N¹⁰-methylpteroyl-glutamic acid(dichloromethotrexate). Other suitable ligands capable of binding tofolate receptors to initiate receptor-mediated endocytotic transport ofthe complex include anti-idiotypic antibodies to the folate receptor. Anexogenous molecule in complex with an anti-idiotypic antibody to afolate receptor is used to trigger trans-membrane transport of thecomplex. Such molecules are used in accordance with the presentinvention as a targeting molecule.

[0053] In a further example of receptor-mediated endocytosis, biotinreceptors have been used to mediate endocytotic activity (Low et al.,1995). Biotin analogs such as biocytin, biotin sulfoxide, oxybiotin andother biotin receptor-binding compounds are ligands that may also beused as suitable targeting molecules to promote the trans-membranetransport of exogenous molecules in accordance with this invention.Other compounds capable of binding to biotin receptors to initiatereceptor-mediated endocytotic transport of the complex are alsocontemplated. These can include other receptor-binding ligands such as,for exmple, anti-idiotypic antibodies to the biotin receptor. Anexogenous molecule complexed with an anti-idiotypic antibody to a biotinreceptor could be used to trigger trans-membrane transport of thecomplex. Such molecules are used in accordance with the presentinvention as a targeting molecule.

[0054] Other examples of targeting molecules include glucose, galactose,mannose, mannose 6-phosphate, hormones (e.g., insulin, growth hormone,and the like), growth factors or cyokines (e.g., TGF-β, EGF,insulin-like growth factor, and the like), YEE(GalNAcAH)₃ orderivatives, cobalamin, α-2 macroglobulins, asialoglycoprotein, albumin,texaphyrin, metallotexaphyrin, antibodies, antibody fragments (e.g.,Fab), single-chain antibody variable region (scFv), transferrin, anyvitamin and any coenzyme.

[0055] As previously described, a bioconjugate of the present inventioncomprises a bioactive agent conjugated directly or indirectly via acovalent bond to the cobalt atom of an organocobalt complex. Thebioactive agent is conjugated directly to the cobalt atom through anon-reactive atom in the bioactive agent or is conjugated indirectly tothe cobalt atom through the use of a spacer. Therefore, in contrast tothe conjugates formed under U.S. Pat. No. 5,428,023, the attachment of abioactive agent to the cobalt atom in the axial position does notinterfere with receptor-mediated endocytosis from the blood into cells.

[0056] The unusually weak cobalt-non-reactive atom bond (e.g., C—Cobond) of the bioconjugate provides a readily addressable trigger for thecontrolled in vivo release of the bioactive agent from the organocobaltcomplex. The bond dissociation energy (BDE) of Co-non-reactive atom bondin the bioconjugate is in the range of 30 to 50 kcal/mol (e.g., 30-40kcal/mol range for a Co—C bond) which make them among the weakestcovalent bonds known, yet the bond is relatively stable in aqueoussolution.

[0057] A common strategy will employ the modification of the anticancerdrug so that it possesses an electrophilic site which can react with thehighly nucleophilic Co(I) intermediate generated upon treatment ofhydroxycobalamin with NaBH₄. This structural modification will besufficiently far removed from the active site (pharmacophore) topreclude any interference with the desired biological activity.Approache used in the case of chlorambucil are typical: the carboxylicacid group of chlorambucil is converted to either an acid chloride or abromoethyl ester, either of which can be efficiently coupled withcob(I)alamin.

[0058] For example, reduced Cbl^(I) is prepared by NaBH₄ or zinc dustreduction, e.g. of hydroxocob(III)alamin. In the above scheme, the drugcan be a cytotoxic agent, other drug or other bioactive agent asdescribed herein. In other schemes, a spacer containing a carbon atom orother atom such as that specified for the non-reactive atom for bindingto the cobalt atom and which also contains a reactive grouping, e.g. —OHor —CN, which is further reacted with the bioactive agent, isintroduced. Other reactive groups, e.g. —NH₂, —SH, —COOH, etc., can alsobe utilized for coupling to a bioactive agent. It is important to notethat, in some cases (e.g., chlorambucil, doxorubicin), the small organicmolecule released is not the parent drug, but rather retains some of themodification installed to allow coupling. In other cases (e.g.,topotecan), the structure of the released drug may correspond to theparent molecule.

[0059] More specific details of the synthesis of representativebioconjugates according to the present invention are as follows, using a“drug” which can be replaced by any suitable bioactive agent andcobalamin which can be replaced by any suitable organocobalt complex. Inthis synthesis, all procedures are under argon. Hydroxocob(III)alamin isdissolved in aqueous CH₃OH (1:1 v/v) at 25° C. A 2-10 fold excess ofNaBH₄ is added. The solution slowly changes color from red to brown andgradually green (Cbll). After approximately 15 min. the electrophilicdrug ligand (dissolved in the same deoxygenated solvent) is added, e.g.,as an alkyl, acyl or aryl chloride. Strictly anaerobic conditions aremaintained and the reaction mixture is stirred gently at 25° C. Thecolor gradually changes back to red as Cb^(I) is converted to alkyl-,acyl-, or aryl-Cbl^(III). After about 1.5 h, the solution is acidifiedto pH 3.0 with dilute HCl. Methanol is removed under reduced pressure byrotary evaporation at less than 40° C. The resulting aqueous solution isdiluted with an equal volume of H₂O and loaded onto a Dowex AG-50-X2(200-400 mesh) cation exchange column. The column is washed sequentiallywith H₂O and 0.1 M NaOAc, pH 6.4. Fractions containingdrug-cob(III)alamins appear red and are collected appropriately.Unreacted hydroxocob(III)alamin is retained on the column. Combinedfractions of drug-cob(III)alamin are extracted with phenol andconcentrated by rotary evaporation. Drug-cob(III)alamins can often becrystallized by the addition of acetone to a concentrated aqueoussolution. Characterization of the alkyl-, acyl-, or aryl-cobalaminconjugates is by NMR, mass spectrometry (FAB, Cl, or electrospray), andIR methods.

[0060] A methotrexate-containing bioconjugate can be synthesized by thefollowing methods.

[0061] In method one utilizing the above procedure, methotrexate (MTX)is converted to its corresponding acyl chloride and reacted withcobalamin and/or Co(III)[SALEN] and/or other disclosed organocobaltcomplexes to yield methotrexate-cobalamin andmethotrexate-Co(III)[SALEN] according to the following reaction schemeI. In the alternative method two, the C—Co bond is first formed from anacyl chloride having a protected amino group. The amino group is thendeprotected, followed by formation of the amide bond to anaminobenzoylpterin according to the following reaction scheme II.

[0062] An aminopterin-containing bioconjugate can be synthesized by thefollowing method. The des-methyl derivative of methotrexate(aminopterin) is coupled to cobalt as shown by the following reactionformula, in which the iminium ion is either reacted with Co(I) directly,or the iminium ion is converted to the aminonitrile and then slowlyunmasked to reveal the iminium cation.

[0063] A topotecan-containing bioconjugate can be synthesized by thefollowing method. The cytotoxic activity of topotecan (TPT) orcamptothecin (CPT) arises from their ability to freeze topoisomeraseI-DNA “cleavable complexes.” (Pommier et al., 1995) Since some tumortypes display greatly elevated levels of topo I (Giovanella et al.,1989), topoisomerase poisons of this type are likely to have a highertherapeutic index in the treatment of those cancers. However, treatmentwith camptothecin derivatives could be made more general if used inconjunction with the targeted delivery approach.

[0064] Topotecan is conjugated to cobalamin, Co[SALEN] and otherorganocobalt complexes according to the following reaction schemes.Camptothecin is conjugated in a similar manner. Preparation of 10a and10b involves similar chemistry to that discussed above for 8a,b.Selective generation of the phenyl chloroformate (25) of topotecan (5)and acylation of Co(I) gives 10a. Exposure of 25 to 18 or treatment of 5with the previously discussed chloroformate 19 furnishes 10b. Conjugates10c,d will require somewhat longer routes, as they cannot be prepareddirectly from 5. However, the established synthetic route for conversionof the natural product camptothecin to 5 can be modified at theappropriate point to allow for attachment of the cobalt complex. Thefirst three steps to prepare phenolic intermediate 26 are known (Mulliezet al., 1994). Mannich-type substitution with formaldehyde/dimethylaminethen gives 5. Use of methylamine gives the corresponding secondary amine27. At this point, linkage to Co via a methylene to give 10c is possiblevia Co(I) trapping of a second, in situ generated imminum salt.Alternatively, N-alkylation with 23 gives 10d. Cleavage of 10a and 10bprovides 5 directly via fragmentative pathway or indirectly via otherproducts. Cleavage of 10c with hydrogen extraction yields 5. Cleavage of10d yields the product 5 having an ethylmethylamino group in place ofthe dimethylamino group.

[0065] A busulfan-containing bioconjugate can be synthesized by thefollowing method. Busulfan is an alkylating agent used therapeuticallyagainst chronic myelogenous leukemia (CML). The preferred point forattaching busulfan to the organocobalt complex is on one of thealkanesulfonate units. A slight change in the structure of the sulfonateportion of the ester is will not exert a large effect on the ability ofthe released drug to crosslink DNA. Cleavage of 7a followed by hydrogenabstraction furnishes the mixed ethanesulfonate/methanesulfonate 2b.Trapping of the carbon radical under oxidative conditions produces mixedbis(sulfonate) 2c, which is also a competent crosslinking agent.Cleavage of 7b results in the release of the parent drug 2a afterhydrogen abstraction. Bis-methylsulfonate busulfan is conjugated tocobalamin, Co[SALEN] and other organocobalt complexes according to thefollowing reaction schemes. For the preparation of 7a, the commerciallyavailable sodium salt of bromoethanesulfonic acid (11) serves as thestarting point. Heating with phosphorus pentachloride furnishes thecorresponding sulfonyl chloride 12 as a distillable liquid. Treatmentwith Co(I) leads to preferential displacement of the bromide to furnish13, which is converted to 7a by sequential treatment with 1,4-butanedioland mesyl chloride. The order of the final three steps can be changed;for example, treatment of 12 with excess butanediol, followed by mesylchloride gives the mixed bis(sulfonate) 14. Selective displacement ofthe primary bromide by Co(I) then gives 7a. In the case of conjugate 7b,treatment of 2-bromobutane-1,4-diol (which is readily available frommalic acid diester) with Co(I) gives adduct 15. Bis(mesylation) gives7b. Alternatively, 7b is prepared from 16 (X═Br or I) with selectivedisplacement of the halide.

[0066] A chlorambucil-containing bioconjugate can be synthesized by thefollowing methods. Chlorambucil is a relatively stable nitrogen mustardwith attenuated alkylating ability, presumably as a consequence of theless-basic aniline nitrogen.

[0067] Method One: In this procedure, chlorambucil is converted to theacid chloride followed by reaction with cob(I)alainin or Co(I)[SALEN]according to reaction sequence I. In situations where the acyl linkageto the organocobalt complex is too labile towards serum nucleophiles,two alternate bioconjugation procedures can be utilized.

[0068] Method Two: The procedure involves bromination of a carbon atomadjacent to the carboxyl group under standard Hell-Vollhardt-Zelinskiconditions to permit attachment of the Co complex in the α-positionaccording to reaction sequence II. In scheme II, replacement of the C—Cowith C-H provides chlorambucil. The reactant stoichiometry, temperature,and dilutions conditions can be manipulated to avoid competingdisplacement of one of the chloroethyl groups, or of the C1 by S_(N)2attack.

[0069] Method Three: The BOC-protected p-aminophenylacetaldehyde can beconjugated to the Co moiety, followed by formation of the activenitrogen mustard product according to the following reaction sequenceIII.

[0070] A chlorambucil, ethyl ester-containing bioconjugate can besynthesized by the following methods. When conjugating a drug via acarboxyl group, as in the case of chlorambucil, linking the drug to thecobalamin via a hydroxyethyl tether may be desirable. This can beaccomplished by one of two convenient routes, both of which areschematically illustrated below. First, 2-hydroxyethyl-cob(III)alamincan be readily prepared from cob(I)alamin and bromoethanol.Esterification is carried out under standard conditions, i.e. byreaction of a carboxylic acid (chlorambucil) with an alcohol(2-hydroxyethylcob(III)alamin) in the presence ofdicyclohexylcarbodiimide (DCC) (or water-soluble derivatives such asEDCI) and a catalytic amount of 4-N,N-dimethylaminopyridine (DMAP) andits hydrochloride salt (DMAP-HCl) in dichloromethane or toluene.Alternatively, the ester-linked conjugate can be prepared by firstforming the 2-bromoethyl ester of chlorambucil and then reacting theester with cob(I)alamin to provide the same product. The reactionschemes (I, II) are shown below. With this mode of attachment, cleavagefrom the bioconjugate leads to release of the ethyl ester ofchlorambucil according to reaction scheme III.

[0071] An etoposide-containing bioconjugate can be synthesized by thefollowing method. Etoposide is a semisynthetic derivative of the naturalproduct epipodophyllotoxin that is widely used against a variety oftumors, especially small cell lung carcinoma and germ cell tumors (DeJong et al., 1995). It has also shown considerable promise in thetreatment of refractory cases of ovarian and breast cancer. Etoposideappears to function as a topoisomerase II poison.

[0072] Etoposide is conjugated to cobalamin, Co[SALEN] and otherorganocobalt complexes according to the following reaction schemes.Bioconjugates 8a and 8b require conversion of the free phenol ofetoposide (3) to the corresponding chloroformate 17. Direct acylationwith Co(I) gives acylCo(III) derivative 8a, while treatment with thepreviously described hydroxyethylCo(III) derivative 18 furnishescarbonate 8b. This derivative is also available via acylation of 3 withthe chloroformate 19 derived from 18. Preparation of acetal-modifiedconjugate 8c may be more challenging. The ethylene acetal of 3 can behydrolyzed and then the acetal reformed using aldehyde 20a or dimethylacetal 20b (Keller-Jusi et al., 1971). Compound 20a may also be accessedvia careful, selective oxidation of 18, while 20b should be availablevia alkylation of the Co(I) derivative with conunerically availablebromoacetaldehyde dimethyl acetal. In addition, the acetal of glucosecan be formed and then the secondary alcohol of 21 can be glucosylated .Cleavage of 8a or 8b either give 3 directly via fragmentative pathways,or furnish products which can undergo eventual hydrolysis to 3. Trappingwith H. following homolysis of 8c would then furnish 3.

[0073] A doxorubicin-containing bioconjugate can be Synthesized by thefollowing method. Doxorubicin (4) is the most widely used of theanthracycline antibiotics, and is clinically useful against a broadspectrum of solid and hematological tumors. Like etoposide, doxorubicinappears to target topoisomerase II, ultimately leading to growth arrestand nonapoptotic cell death (Fornari et al., 1996; Ling et al., 1996).The clinical usefulness of doxorubicin is limited by nonspecifictoxicity, especially cardiotoxicity. Thus, it would appear to be aparticularly good candidate for selective delivery. This is confirmed byits frequent use in liposome-based methods (Hu et al., 1996; Longman etal., 1995; Hosada et al., 1995), as part of immunoconjugates (Johnson etal., 1995; Sivam et al., 1995), or in prodrug approaches (Svensson etal., 1995).

[0074] Doxorubicin conjugated to cobalamin, Co[SALEN] and otherorganocobalt complexes according to the following reaction schemes. Forthe synthesis bioconjugate 9a, the condensation of the daunosamine aminogroup with acyl-Co(III) complex 22 is performed. This reaction forms the2-pyrroline ring in analogy to published routes using4-iodobutyraldehyde and 5-iodo-2-pentanone.(9b) The acyclic tertiaryamine derivative 9b is available from 4 via initial reductive aminationwith acetaldehyde, then alkylation of the resulting secondary amine withthe mesylate 23 derived from 18. Alternatively, treatment of 4 withchloroformate 19 provides carbamate 9e. If alternative points ofattachment are desired, hydrazone-linked derivatives such as 9d can beused using simple cobalamin alkyl hydrazides such as 24, obtainable from23. The cleavage of these bioconjugates is shown in the reaction schemebelow.

[0075] A carboplatin containing bioconjugate can be synthesized as shownin the following reaction scheme.

[0076] A peptide-containing bioconjugate can be synthesized by thefollowing methods: (1) The C-terminal carboxyl group of the peptide canbe activated and used to acylate Co(I), in analogy to the acylation ofCo with chlorambucil acid chloride. (2) The C-terminal carboxyl groupcan be esterified with bromoethanol, in analogy to the otherchlorambucil route, and the bromide displaced with Co(I). (3) TheN-terminal amino group of the peptide can be treated with CH₂═O andCo(I) to form a Co(III)—CH₂—NH-peptide linkage, in analogy to thesynthesis of the topotecan bioconjugate. (4) A Co(III)-amino acidcomplex can be prepared, and used in a coupling step with the remainderof the peptide. These methods can involve attachment of the Co at eitherthe N- or C-terninus, or via a side-chain. A longer linker may beemployed in any of these routes, if it is desirable to keep the cobaltcomplex further removed from the peptide chain.

[0077] An oligonulceotide or nucleic acid containing bioconjugate can besynthesized by the following methods. In both methods, a phosphate esteris used to link the end of the nucleotide and a hydroxyethyl-Co group.This linkage can be accomplished by either directly couplingCo—CH₂CH₂—OH and Nucl-OPO₃ ²⁻, or by esterifying Nucl—OPO₃ ²⁻ withBr—CH₂CH₂—OH, then displacing the Br with Co(I), as above.

[0078] An unsymmetrically substituted Co[SALEN] complex can be preparedfrom 5-amino-salicylaldehyde and the glycolate ether of2,5-dihydroxybenzaldehyde. The amino group, which is prepared fromcommercially available 5-nitrosalicylaldehyde, functions as theattachment for the targeting molecule (binding domain, BD) by way ofEDCI-catalyzed amide formation. The other molecule has a carboxylic acidunit attached for solubility enhancement. Coupling of these twomolecules with ethylenediamine and Co(II) acetate furnishes a mixture ofthree comlexes: the two symmetrical complexes and the mixed one. All ofthese are useful, although the one lacking a BD-unit attached to eitherside of th e SALEN is less preferred.

[0079] With regard to binding domains, two possiblities are shown: acobalamin derivative, and a peptide. In the former case, the knowncarboxylic acid i s u sed to attach cobalamin to the amino group of theSALEN. This bioconjugate still usees cobalamin-based receptor-mediatedendocytosis to get into the cell, but the drug is attached through theSALEN instead of the cobalamin. The latter case uses a peptide known tobind to cell surface receptors of tumor cells (e.g., a fragment ofepidermal growth factor), with the carboxyl terminus attached to theamino group on the SALEN. Alternatively, one of the glutamate carboxylgroups of folate is used to obtain a folate-based bioconjugate. Inaddition to connecting the binding domain via an amide linkage, onecould use reductive amination if the targeting molecule contained analdehyde (BD-CHO+SALEN-NH₂+NaBH₄), or one could use the carboxyl groupon the other piece to form an amide or ester linkage. Many otherapproaches (e.g., ether formation, olefination by Wittig reaction,attachment via a diester or diamide linker, etc.) are also possible.

[0080] Extended benzenoid systems of the SALEN ligands are shown below.

[0081] As a starting point in their synthesis, any of the commerciallyavailable naphthalene diols can be used. The diols undergo formylationreactions to Furnish the molecules shown below. These moleucles are thenbe coupled with Co(II) acetate and various diamines to give the extendedCo[SALEN] complexes. The OH groups on the second ring can be leftintact, used to attach the binding domain, or modified to enhance watersolubility through attachment of a polar group, such as polyamine,polycarboxylic acid, or carbohydrate moiety.

[0082] Modification of quinolines and isoquinolines are also carried outto give pyridine-fused SALENs.

[0083] Along similar lines, SALENs derived from monocyclic heteroyclichydroxyaldehydes are made, examples of which are shown below.

[0084] A bioconjugate containing the “green corrinoid” (Brown et al.,1996) can be synthesized as follows. The “green corrinoid” can bereduced in analogy to cobalamin, and that the reduced corrinoid willreact with iodomethane to form the methylCo(III) corrinoid. Since themethylCo(III) corrinoid exhibits similar behavior to natural cobalamin,similar conjugation procedures with the electrophilic drug derivativesdescribed above can be carried out.

[0085] The bioconjugates of the present invention have the improvedproperty of being capable of targeted, selective release of thebioactive agent from the bioconjugate. The bioactive agent is isreleased from the bioconjugate by cleavage. In one embodiment, thecleavage may occur as a result of normal displacement by cellularnucleophiles or enzymatic aciton. In a second embodiment, the cleavageis caused to occur selectively at the release site by an externalsignal. The external signal may be light or photoexcitation, i.e.photolysis, or it may be ultrasound, i.e. sonolysis. Further, if thephotolysis takes place in the presence of a magnetic field surroundingthe release site, the release of the drug, such as a cytotoxic agent,into surrounding healthy tissue can be minimized.

[0086] Although it is desired to cause the bioactive agent to bereleased at the desired cells, tissue or organs, e.g., at the site ofthe tumor or other cancer cells, it is also desirable to protectadjacent tissues from the negative side effects of such potent agents.The bioactive agent is released from the bioconjugate at the targetedsite preferably by application of an external signal, such as light orultrasound. The photolysis of the bioconjugates of the present inventionoccurs though cleavage of the Co—C bond to produce a solvent-cagedradical pair consisting of Co(II) and the bioactive agent radical (R.).Lott et al. (1995) demonstrate that alkylcob(III)alamin photolysis canundergo magnetic field dependent recombination. A magnetic fieldapplication of 100-3000 gauss can be used to enhance radical pairrecombination in surrounding tissue where drug release from theconjugate is not desired, leading up to at least a 2-fold decrease inphotochemically-triggered drug release in such surrounding healthytissue.

[0087] The sonolysis of the bioconjugates of the present inventionoccurs through cleavage of the Co-non-reactive atom bond in aqueoussolution to produce the bioactive agent and a Co(II) (e.g.,cob(II)alamin (Cbl^(II))) under anaerobic conditions or the bioactiveagent and aquoCo-(III) (e.g., aquocob(III)alamin (H₂O—Cbl^(III))) underaerobic conditions. In either event, sonolysis from the focusedapplication of ultrasound, results in Co-non-reactive atom bond cleavageand the irreversible release of the bioactive agent from theorganocobalt complex.

[0088] The bioconjugates of the present invention can undergo naturalcleavage as follows. Bioconjugates may be cleaved by natural means suchas by serum nucleophiles. Once inside the cell, cobalamin-drugbioconjugates (utilized here only as an example and not intended tolimit the invention) can undergo cleavage by a variety of mechanisms.Standard B₁₂ ligand exchange mechanisms permit the displacement of thedrug. Alternatively, cellular nucleophiles can attack at either thecarbon or the cobalt atoms of the Co—C bond. Cyanide is the most commonexample of a nucleophile which attacks at cobalt, leading tocyanocob[III]alamin and a free drug in which the former C—Co bond hasbeen replaced with a C—H bond. Thiols (such as are found in cysteine orglutathione) can attack at carbon, leading to a reduced cob[I]alamin anda free drug which incorporates the former thiol group. (e.g.,R—Co[III]+R′—SH+base→R—S—R′+Co[I]+base-H.) Hydroxide and other basicagents can also cleave organic ligands from Co[III] complexes, althoughthis typically occurs via an elimination process which alters thestructure of the ligand through incorporation of a new double bond. Inaddition, B₁₂ metabolic enzymes present in cells can result in thecleavage of the bioactive agent from the co-atom.

[0089] The bioconjugates of the present invention can undergo cleavageby photoactivation or photolysis as follows. The photochemical releaseof the bioactive agent from the organocobalt complex can be triggered bythe application of visible light at 400-800 nm. It is preferred to useorganocobalt complexes which require longer wavelengths of visible light(600-800 nm), more preferably red light (600 to 750 nm). When photolysisis utilized for the release of the bioactive agent form thebioconjugate, it is particularly preferred that the non-reactive atom ofthe bioactive agent or the atom of the spacer bound to the cobalt atombe a carbon atom.

[0090] The vitamin B₁₂ cofactor occurs naturally in two forms:adenosylcob(III)alamin, (AdoCbl^(III)), also known as coenzyme B₁₂, andmethylcob(III)alamin (CH₃Cbl^(III)). The remarkably weak C—Co bond fromthe corrin ring to the 5′-deoxyadenosyl or methyl ligand imparts a mostunusual chemistry to cobalamins. The C—Co bond energy in AdoCbl^(III)and CH₃Cbl^(III) is estimated to be as low as 31 and 37 kcal/mole,respectively. This makes the C—Co bond one of the weakest covalent bondsknown and allows photocleavage of the bond by visible light. The initialproduct of the photocleavage of AdoCbl^(III) is the geminate radicalpair {CH₂-Ado : Cbl^(II)}. Brackets {: } indicate the radical pair isgeminate (born of the same parent molecule) and held in close proximityby solvent interactions. Picosecond laser flash photolysis experimentsof AdoCbl^(III) have shown this to be a reversible process with ageminate recombination rate constant of k_(rec)≈1×10⁹ s⁻¹, followingphotolysis. Recent nanosecond laser flash photolysis studies have probeda slower radical pair recombination that occurs in the solvent and islimited by diffusion.

[0091] The π-π* electronic transition of the corrin ring of cobalaminproduces a long wavelength absorption maximum at 525 nm, as shown byFIG. 1. Irradiation of alkylcobalamins at this wavelength leads tocleavage of the C—Co bond with a photolysis quantum yield of 0.1-0.3.The in vivo photolysis of bioconjugates according to the presentinvention is preferably accomplished by delivering the light with afiber optic probe because of the strong absorption of hemoglobin nearthis wavelength. To avoid potential problems with the absorptionspectrum of cobalamin while maintaining a photolabile C—Co bond,Co[SALEN], which is a five-coordinate analogue of coenzyme B₁₂, can beused. Alkyl-Co[SALEN] complexes have absorption maxima near 650 nm, withsignificant absorption beyond 700 nm as shown by FIG. 2. This is adistinct advantage for photoactivatable drug release, as human tissuebecomes increasingly transparent above 610 nm. Other synthetic ornaturally occurring B₁₂ derivatives are, or may become, available thathave absorption maxima above 600 nm. For example, a green cobaltcorrinoid having an absorption maximum at about 624 nm is reported byBrown et al. (1996). Human tissue becomes transparent to depths of up toabout 5.7 cm at wavelengths of between about 600 and 800 nm. The use oflonger wavelengths enables the more selective irradiation of limitedportions of a subject's body, with consquent release in a small targetregion.

[0092] The bioconjugates of the present invention can undergo cleavageby photoactivation or photolysis in the presence of a magnetic field asfollows. The use of the magnetic field further limits the release of thebioactive agent to the desired site. For example, because of thetoxicity of antineoplastic agents to healthy tissues, it is incumbentupon any effective site specific delivery system to limit damage tocells other than at the site of activity. In the photohomoylsis cleavagereaction, the electrons in the broken covalent bond start out with theirspins paired ↑⇓ (singlet state) from having participated in the covalentbond. During the early lifetime of the radical pair, the spins retaintheir original orientation R↑+⇓R′ until the electron spins randomizeover time. During the time the spins are paired, the radicals canrecombine and revert to the starting material. If either of these pairedradical spins should intersystem cross (ISC) to the triplet spin state(R↑+⇓R′) they can no longer recombine until their spins are once againpaired. That situation is preferred to release the bioactive agent fromthe bioconjugate. However, in healthy tissue, it is desired to prevent,or at least minimize, the cleavage of the conjugate. It that event, itis desirable to alter the rate of ISC by introducing an externalmagnetic field that increases the gap between the triplet state energylevels, thus encouraging the recombination of the original bioconjugatein the healthy tissues.

[0093] The recombination rate can be increased by application of amagnetic field in the range of 100-3000 gauss to the healthy tissuesleading to a net decrease in the photochemical quantum yield and adecrease in drug release into healthy tissues by a factor of at least 2.Application of about 300 to 1000 gauss is considered to be optimal. See,Grissom (1995).

[0094] The bioconjugates of the present invention can undergo cleavageby ultrasound or sonolysis as follows. Although any non-reactive atomcan be bound to the cobalt atom in the bioconjugate and cleaved bysonolysis, it is preferred that the atom be a carbon atom. The vitaminB₁₂ cofactor occurs naturally in two forms: adenosylcob(III)alamin,(AdoCbl^(III)), also known as coenzyme B₁₂ and methylcob(III)alamin,(CH₃Cbl^(III)). The remarkably weak C—Co bond from the corrin ring tothe 5′-deoxyadenosyl or methyl ligand imparts a most unusual chemistryto cobalamins. The C—Co bond energy in AdoCbl^(III) and CH₃Cbl^(III) isestimated to be as low as 31 and 37 kcal/mole, respectively. This makesthe C—Co bond one of the weakest covalent bonds known and allowssonolysis of the bond by the application of ultrasound in the range ofabout 20 kHz-500 MHz, preferably 20 kHz-100 MHz, more preferably 20 kHzto 10 MHz.

[0095] Sonolysis of aqueous solutions produces a high concentration ofhydroxyl radicals and hydrogen atoms according to the equation:

H—OH→H.+.OH

[0096] These reactive oxidizing and reducing species are responsible forinitiating most reactions in aqueous solvents. Ultrasound irradiationand sonochemistry are often not described as high-energy processes, butduring sonolysis, the development, growth and implosion of bubbles in aliquid create extreme reaction environments on a microscopic scale. Thecollapse of cavitation bubbles produces pressures >500 atm. andtemperatures >5000° C. The radicals formed survive the collapse of thebubbles. The formation of hydroxyl radicals in vivo has been the focusof several investigations because of the potentially deleterious effectsof oxidizing free radicals in human tissue. However, such radicals donot present an unacceptable health risk, as clinical experience hasdemonstrated that diagnostic ultrasound is a benign procedure. Theability to form these radicals by sonolysis in vivo provides a mechanismfor the triggered release of active neoplastic and other agents fromconjugation with B₁₂, Co[SALEN] or other suitable cobalamin or cobalaminlike substrates.

[0097] In the sonolysis of drug-B₁₂ conjugates (utilized here only as anexample and not intended to limit the invention), the C—Co bond iscleaved in aqueous solution to produce the drug and a cob(II)alamin(Cbl^(II)) under anaerobic conditions or the drug and aquocob(III)alamin(H₂O-Cbl^(III)) under aerobic conditions. The cleavage is not a directbreaking of the C—Co bond. Rather, under anaerobic conditions thepredominant pathway for C—Co bond cleavage by sonolysis is throughreduction of drug-Cbl^(III) to the labile drug-Cbl^(II) by H., followedby dissociation to the closed-shell drug and Cbl^(II). The reaction ofHO. with drug-Cbl^(II) leads to H₂O—Cbl^(III), as well as degradation ofthe corrin ring. Under aerobic conditions, the pathway for the C—Co bondcleavage by sonolysis is through reduction of the drug-Cbl^(III) toproduce drug and Cbl^(II), but O₂ oxidizes Cbl^(II) to H₂O—Cbl^(III). Ineither event, sonolysis from the focused application of ultrasound,results in C—Co bond cleavage and the irreversible release of the drugfrom the cobalamin. Therefore, sonolysis-triggered release can occurunder aerobic and hypoxic conditions alike.

[0098] The present invention is useful in the treatment of (including,but not limited to) cancer, hepatitis, psoriasis and other localizeddiseases, as well as for gene therapy and peptide therapy. Thebioconjugates according to the present invention can be administered byany route, including intravenously, parenterally, orally,intramuscularly, intrathecally or as an aerosol. The mode of deliverywill largely depend on the biological effect desired to be achieved. Askilled artisan will readily know the best route of administration for aparticular treatment in accordance with the present invention. Theappropriate dosages will depend on the route of administration and thetreatment indicated, and can be readily determined by a skilled artisan,such as by extrapolation from current treatment protocols. If theorganocobalt complex of the bioconjugate is cobalamin or derivative oranalogue, it is preferred to administer orally a bolus of vitamin B₁₂prior to administration of the bioconjugate to reduce or eliminatepotential hepatotoxicity which might otherwise result from theadministration of the bioconjugate. The oral dose of B₁₂ will saturatethe enterohepatic shuttle system and load hepatocytes with cobalamin. Itis preferred that 0.1 mg to 100 mg, more preferably 1. 0 mg to 10 mg, ofvitamin B₁₂ be administered prior to the administration of thebioconjugate containing cobalamin. In addition, vitamin B₁₂ can beadministered, preferably intravenously, following the selective cleavageof bioconjugate to wash out all bioconjugate which has not been cleaved,and thus further reduce potential systemic effects. It is preferred that0.1 mg to 100 mg, more preferably 10 mg to 100 mg, of vitamin B₁₂ insaline be administered intravenously over 4-5 hrs. Finally, prior to theadministration of a cobalamin-based bioconjugate, nitrous oxide can beadministered to the subject in order to deplete body stores of cobalaminin its various forms, such as methylcobalamin. Administration of nitrousoxide has the effect of creating a greater body deficit of cobalaminbefore administration of the cobalamin-based bioconjugate.

[0099] Pharmaceutical compositions containing a compound of the presentinvention as the active ingredient can be prepared according toconventional pharmaceutical compounding techniques. See, for example,Remington's Pharmaceutical Sciences, 17th Ed. (1985, Mack PublishingCo., Easton, Pa.). Typically, an antagonistic amount of activeingredient will be admixed with a pharmaceutically acceptable carrier.The carrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., intravenous, oral,parenteral, intrathecal, transdermal, or by aerosol.

[0100] For oral administration, the compounds can be formulated intosolid or liquid preparations such as capsules, pills, tablets, lozenges,melts, powders, suspensions or emulsions. In preparing the compositionsin oral dosage form, any of the usual pharmaceutical media may beemployed, such as, for example, water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents, suspending agents, andthe like in the case of oral liquid preparations (such as, for example,suspensions, elixirs and solutions); or carriers such as starches,sugars, diluents, granulating agents, lubricants, binders,disintegrating agents and the like in the case of oral solidpreparations (such as. for example, powders, capsules and tablets).Because of their ease in administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe sugar-coated or enteric-coated by standard techniques. The activeagent must be stable to passage through the gastrointestinal tract. Ifnecessary, suitable agents for stable passage can be used, and mayinclude phospholipids or lecithin derivatives described in theliterature, as well as liposomes, microparticles (including microspheresand macrospheres).

[0101] For parenteral administration, the compound may dissolved in apharmaceutical carrier and administered as either a solution or asuspension. Illustrative of suitable carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal,vegetative or synthetic origin. The carrier may also contain otheringredients, for example, preservatives, suspending agents, solubilizingagents, buffers and the like. When the compounds are being administeredintracerebroventricularly or intrathecally, they may also be dissolvedin cerebrospinal fluid.

[0102] In the treatment of cancer (sarcomas, carcinomas or leukemias), adistinction can be made between the types of systemic adjuvantchemotherapies that are typically used in concert with the more extrememethods of surgical excision and radiation therapy. There are two broadclasses of chemotherapeutic adjuvants: (1) endocrine and antivasogenictherapeutic agents which are aimed at altering the body's physiology;and (2) cytotoxic chemotherapeutic agents which are typicallyadministered systemically to kill or inhibit the growth of transformedcells.

[0103] Cytotoxic or antineoplastic agents are represented by a number ofdrug classes. Alkylating agents undergo chemical reactions that generatehighly reactive electrophilic carborationsnium ions that readily formcovalent linkages (alkylate) with various nucleophilic biologicallyimportant moieties such as nucleic acid bases and phosphate, amine,sulfhydryl, hydroxyl, carboxyl and imidazole groups. These agents, amongother functions, have cytotoxic actions that disturb the fundamentalmechanisms concerned with cell growth, mitotic activity, differentiationand function. Chlorambucil, modified busulfan, cyclophosphamide,ifosfamide and cisplatin and its structural analogs are representativealkylating agents.

[0104] Antimetabolites such as folic acid analogs (e.g. methotrexate)and pyrimidine analogs (e.g. fluorouracil and fluorodeoxyuridine) exertcytotoxic activity by blocking or preventing metabolic pathways leadingto neoplastic cell destruction. Methotrexate is also known to be usefulin the treatment of psoriasis by inhibiting the proliferation ofepidermal cells.

[0105] Another potent cytotoxic class is mitotic inhibitors such as thepaclidaxel or the alkaloids camptothecin, vincristine and vinblastine.

[0106] Also, certain antibiotics, such as doxorubicin and daunorubicin,(tetracyclic aglycone glycosides), intercalcate with DNA and inhibitnucleic acid synthesis.

[0107] In accordance with the present invention, bioconjugates for thetreatment of cancer are formed preferably using chemotherapeuticsselected from the group consisting of alkylating agents, antimetabolitesand mitotic inhibitors. For example, methotrexate is an antimetabolite;chlorambucil, cisplatin and modified busulfan are alkylating agents, andcamptothecin and its derivatives are alkaloids. The bioconjugates formedfrom these cytotoxic agents can be administered intravenously for thetreatment of the specific classes of cancer for which they are known tobe effective, e.g. cancer of the colon, lung, kidney, breast, prostate,melanoma, nasopharyngeal, T-cell leukemia, myelogenous leukemia,lymphocytic leukemia and the like. When delivered intravenously to theblood stream and the bioconjugates contain cobalamin, the naturalaffinity of cancer cells for B₁₂ will target the bioconjugates to thesetissues or cell sites. Alternatively, the bioconjugates can beengineered to be selective for the delivery of the chemotherapeuticagent to the desired cancer cell by the incorporation of a suitabletargeting molecule (such as those set forth above) on the organocobaltcomplex.

[0108] In accordance with the present invention, solid tumors aretreated as follows, with use of a drug-B₁₂ bioconjugate as an example.This example is not intended to limit the present invention in anymanner, and a skilled artisan could readily determine otherbioconjugates of the present invention which could be utilized for thetreatment of solid tumors. The drug-B₁₂ bioconjugate is administered,preferably intravenously, to a cancer patient to target metastaticcancer when the cancer cell has a significant requirement for cobalamin.This propensity of cobalamins to migrate to the cancer cellssignificantly reduces cardiotoxicity, myelotoxicity, hepatotoxicity andsimilar side effects that limit the size and frequency of effectivedosing of antineoplastic agents. Furthermore, problems associated withtoxicity to non-targeted cells is minimized. Delivery is furtherenhanced by the triggering of the release of the antineoplastic agentfrom the bioconjugate by the mechanism of photolysis or sonolysis whichprovides for a high degree of spatial and temporal control of the drugrelease at a localized area over a short time. The application of amagnetic field with photolysis further serves to protect health cells byrecombination of the bioconjugate and limit the release of active agentto the specific cancer cell-containing site(s).

[0109] Although chemotherapy is generally reserved for targetingmetastasized cells after the surgical excision of the primary tumormass, the triggered release of a bioactive agent drawn to the tumor siteallows for treatment of the primary tumor, as well as metastaticneoplasms that have spread to a limited and known area. The bioconjugatedosage, length of treatment, degree of photoactivation, and othertreatment parameters can be determined by one skilled in the art basedon the type of cancer, antineoplastic agent administered, specificcobalamin used, condition of the patient and other factors which arevariable and best determined on a case-by-case basis.

[0110] In accordance with the present invention, leukemia is treated asfollows, with use of a drug-B₁₂ bioconjugate as an example. This exampleis not intended to limit the present invention in any manner, and askilled artisan could readily determine other bioconjugates of thepresent invention which could be utilized for the treatment of leukemia.At least two forms of leukemia, chronic myeloid leukemia (CML) and acutepromyeloctyic leukemia (APL), produce high levels of B₁₂ bindingproteins that result in a 3- to 36-fold increase in the unsaturated B₁₂binding capacity in the blood. The increased concentration of B₁₂binding proteins is consistent with the rapid turnover of immature bloodcells and provides an opportunity to target the delivery of antileukemicdrugs, such as bis-alkylating agents derived from busulfan, to thetransformed hematopoietic cells responsible for the leukemic condition.The bioconjugates of this invention provide a means for the effectiveadministration of such alkylating agents to cell sites from which theactive agent can be released from the conjugate. This targeted deliveryand release provides a significant advance in the treatment of CML andAPL, for which current chemotherapy methods sometimes provide incompleteremission.

[0111] The present invention is also useful for the treatment ofpsoriasis. Psoriasis is a prime target for the transdermally or orallycontrolled delivery of antimetabolites activated by photolyticallyinduced cleavage. Although not life-threatening, psoriasis cansignificantly diminish the quality of life of patients who experiencesevere exfoliation associated with psoriatic and rheumatoid arthritis.Antimetabolites, such as methotrexate and 5-fluorouracil, are effectivein controlling severe cases of skin proliferation. Effective oralTherapy is limited by hepatotoxicity in spite of low dosing, and therisk of cumulative liver damage requires such therapy to be reserved foronly the most severe episodes during a patient's life. The delivery ofsuch agents to the skin improves the appearance and psychologicalquality of life of patients whose lives have been dominated by severepsoriasis.

[0112] The present invention is further useful for peptide therapy. Oneexample of peptide therapy is as a cytotoxic agent, for example as anantineoplastic agent. In this example, a bioconjugate containing theenzymatic domain of diphtheria toxin (DT) is administered to a subjectsuch as described above for solid tumors or leukemia. The targetedrelease of the DT peptide results in the inhibition of protein synthesisand eventual cell death.

[0113] The present invention is also useful for gene therapy. Oneexample of gene therapy is the delivery of an antisense oligonucleotideto inhibit viral gene expression and viral replication. In this example,a bioconjugate containing an antisense oligonucleotide against hepatitisB virus is administered to a patient having a hepatitis B infection. Theaccumulation of the bioconjugate and release of the antisenseoligonucleotide in the liver inhibits hepatitis B virus gene expressionand replication.

[0114] The present invention is further described by reference to thefollowing Examples, which are offered by way of illustration and are notintended to limit the invention in any manner. Standard techniques wellknown in the art or the techniques specifically described below wereutilized.

EXAMPLE 1 Photolysis of B₁₂ and Co[SALEN] Bioconjugates

[0115] A protocol for a typical anaerobic continuous-wave photolysis ofmethylcob(III)alamin or CH₃Cbl^(III) is as follows. An aqueous solutionof 200 μM CH₃Cbl^(III) and 50 mM K⁺ Hepes pH 7.3 is placed in a 1 cmquartz cuvette. Samples that are to be photolyzed under anaerobicconditions are either subjected to repeated freeze-pump-thaw cycles, orpurged with Ar for 40 minutes immediately prior to photolysis andsealed. Continuous-wave visible light irradiation is accomplished at thedesired wavelength with an Ar⁺ pumped dye laser. The incident light onthe face of the cuvette is reduced to 12 mW cm⁻² with neutral densityfilters. The light flux is determined by potassium ferrioxalateactinomety and by a Scientech surface-reading thermopile. The cuvette isplaced in a thermostated cell holder at 25-37° C. For quantum yieldmeasurements as a function of magnetic field, the cuvette is placed inthe gap of a GMW Associates electromagnet with 7.5 cm diametercylindrical poles. The magnetic field within the area of the cuvette ishomogeneous to within 2% and the long term stability is better than 0.5%as monitored by a transverse Hall probe and digital teslameter.

[0116] Absorption spectra from 300-600 nm are recorded in one secondwith a diode array spectrophotometer at variable time intervals from 10seconds to 2 minutes (depending upon the fluence of the photolyzinglight source) for a total of 3τ_(½). Exposure to light during analysisis kept to a minimum. The concentration of CH₃Cbl^(III) is determinedusing the measured absorbance at 350 or 520 nm by the method of Chen andChance (1993). The plot of [CH₃Cbl^(III)] vs. time (t) appearszero-order in all cases.

[0117] Selection of Photolysis Wavelength:

[0118] The absorption spectra for CH₃Cbl^(III) and ethyl-Co[SALEN] areshown in FIGS. 1 and 2. For CH₃Cbl^(III) the π-π* electronic transitionsthat lead to cleavage of the C—Co bond are maximal at 377 and 528 nm.Much preliminary work with B₁₂ photolysis has been carried out with 514nri light from an Argon-ion (Ar⁺) laser. This is close to thelong-wavelength maximum absorbance and gives a quantum yield of about0.3 for CH₃ Cbl^(III).

[0119] The absorbance of blood and tissue is significant at this optimalwavelength for cob(III)alamin excitation. Blood has a low (partial)transmittance window near 514 nm. This absorbance is sufficient toquickly pyrolize whole bovine blood placed in the light path of a 20W/cm² beam of 514 rim light.

[0120] It would therefore be beneficial to provide a cobalamin forconjugation wherein the π-π* electronic transitions that lead tocleavage of the C—Co bond are maximal at a wavelength where there isminimal or no interference. Above about 610 nm blood becomes partiallytransparent and losses beyond 50% transmittance are largely due to lightscattering from the erythrocytes. Heparinized bovine blood placed in thelight path of a 20 W/cm 2 beam of 630 nm light shows only minor heatingover long exposure times. There is also demonstrated a hightransmittance of tissue at 610-800 nm.

[0121] This suggests the use of an organocobalt complex for conjugationhaving an absorption wavelength where tissue and blood are relativelytransparent. FIG. 2 shows that ethyl-Co[SALEN] complexes have absorptionmaximums near 650 nm, with significant absorption beyond 700 nm. AnAr⁺-pumped dye laser or a Krypton-ion (Kr⁺) laser can be a suitablehigh-intensity source of photons in the region of 610 nm. Ar⁺-pumped dyelasers are often used for photodynamic therapy with hematoporphyrins.Also, an inexpensive He—Ne laser, having a principal line at 633 nmmight be used. However, such lasers are typically limited to 50 mWmaximum output.

[0122] There are laser dyes in the 600-700 nm region that can achieveenergy conversion efficiencies of up to 45%. This means that a 6 W Ar⁺pump laser can yield nearly 3 W of spatially-coherent monochromaticlight in the region of 610-750 nm. The exact wavelength can be chosen tooptimize the continuous-wave quantum yield and still maintain areasonable degree of tissue penetration. In tests with alkyl-Co[SALEN]complexes it has been found that 690 nm light from an Ar⁺-pumped dyelaser operating with rhodamine 6-G dye is satisfactory. Optimization canbe determined depending on the specific cobalamin chosen for animaland/or clinical trials of the bioconjugates. In addition, high-powerdiode lasers that emit red light of the desirable wavelength arecommercially available. These diode lasers have the advantage ofproviding up to 100 watts of optical power in a narrow region of theoptical spectrum that is useful for triggering cleavage of thebioconjugates.

EXAMPLE 2 Sonolysis of B₁₂ and Co[SALEN] Bioconjugates

[0123] Sonolysis was carried out with a Branson ultrasonic bath (model3200) operating at 47 kHz. The correct placement of the reaction vesselat a focal point of high-intensity ultrasound was determined by theoxidation of iodide to iodine in the presence of starch (Mason, 1991)and the temperature of the bath was maintained at 21° C. by a constanttemperature circulator. Aerobic sonolysis was typically carried out in atest tube or Erlenmeyer flask, whereas anaerobic sonolysis was carriedout in a closed reaction vessel fitted with a sidearm and quartzcuvette. Anaerobic conditions were produced by sparging with Ar for 30min prior to sonolysis. In some experiments, the pH was buffered by theuse of 100 mM phosphate (aerobic experiments) or 100 mMN-(2-hydroxyethyl)piperazine-N-2-ethanesulfonate (Hepes) (anaerobicexperiments), as specified. All procedures were carried out in theabsence of light to prevent photolytic cleavage of the Co—C bond.Absorption spectra were recorded on a diode array spectrophotometer (HP8452A). The solutions were transferred to a quartz cuvette with a 1 cmlight path for all optical measurements and care was exercised to ensurethat insignificant photolysis occured during the 1 s measurement time.

[0124] Sonolysis of Methylcob(III)alamin:

[0125] Sequential absorption spectra of aqueous CH₃—Cbl^(III) as afunction of anaerobic sonolysis (pH 7.38, 100 mM Hepes, saturating Ar)are shown in FIG. 3A. The absorbance at 340, 374, and 520 nm decreaseslinearly as a function of sonolysis time and the absorbance at 316 and420 nm increases linearly, thereby indicating the reaction is zero orderin substrate concentration. The isosbestic points at 336, 390, and 585nm are in agreement with those obtained through anaerobic photolysis ofCH₃—Cbl^(III). Under the conditions of sonolysis, an additionalisosbestic point occurs at 476 nm, rather than at 486 nm, as typicallyobserved in the course of photolysis. This slight shift in theisosbestic point is caused by a minor product that has an absorbancemaximum near 490 nm. This may be cob(I)alamin that has a sufficientlifetime (t_(½)=22 min at pH 6) to be observed spectrophotometrically.The absorption band at 374 nm is characteristic of a C—Co bond, and itsdisappearance unambiguously indicates displacement of the axial carbonligand.

[0126] Under aerobic conditions, molecular oxygen scavenges H. andprevents the reduction of CH₃—Cbl^(III) via the equation

O₂+H.→.OOH

[0127] In the absence of an organic buffer with abstractable hydrogenatoms, reaction via HO. remains to be a viable process.

[0128]FIG. 3B shows the change in absorbance spectra following aerobicsonolysis in the absence of organic buffer. The decrease in absorbanceat 340 and 374 nm is linear with increasing sonolysis time indicatingthe reaction is zero order in substrate concentration, but theunexpected increase in absorbance at 520 nm indicates the stable productof cobalamin sonolysis is not hydroxocob(III)alamin, as would beexpected if molecular oxygen were to reoxidize cob(II)alamin tocob(III)alamin. Aerobic photolysis under the same conditions shows theexpected decrease in absorbance at 374 nm but no change at 520 nm. Thisdifference suggests that HO. is able to displace the alkyl ligand fromCol^(III), but other HO. reactions also occur (perhaps through thesecondary products HOO. and .O₂ ⁻) to oxidize the corrin ring. Similarabsorbance spectra are obtained from sonolysis of an aerated aqueoussolution containing 100 mM phosphate buffer.

[0129] A similar result is seen in the reaction of CH₃—Cbl^(III) with H.and HO. when these radical species are generated by pulse radiolysis(Blackburn et al., 1972). Reducing species H. reacts to produce the samespectral changes as shown in FIG. 3A. Multiple oxidizing species (HOO.and .O₂ ⁻) can react with CH₃—Cbl^(III) to cleave the Co—C bond, butthese species also lead to the irreversible degradation of the corrinring, as evidenced by the spectral changes similar to those seen in FIG.3B. A precedent for irreversible oxidation of the corrin ring exists inthe photooxygenolysis of alkylcobalamins by singlet oxygen (Krautler andStepanek, 1985).

[0130] Aerobic sonolysis of solutions containing 100 mM Hepes or 100 mMt-butyl alcohol produces no change in the absorption spectra overcomparable time. This is because molecular oxygen quenches the H.reaction pathway, and t-butyl alcohol quenches the HO. reaction pathway.Although Hepes has not previously been reported to be a scavenger ofHO., many reports indicate that organic solute molecules such asformate, can inhibit the reaction of HO. (Weissler, 1962). The absenceof any spectral changes under these conditions suggests that directsonolysis of the Co—C bond is not an important reaction pathway.

EXAMPLE 3 Biological Testing Against NCI Human Tumor Cell Lines

[0131] The efficacy of the bioconjugates is tested against tumor celllines using existing protocols for assessing the effectiveness oftargeted coenzyme B₁₂ antineoplastic agent-containing bioconjugates.Representative cell lines tested include HCT 116 (human colon tumor),A549 (human lung), ACHN (human kidney), MCF7 (human breast), humanprostate, SK5-mel (human melanoma), KB (human nasopharyngeal), CCRF-CEM(human T-cell leukemia), HL-60 (human promyelocytic leukemia), RD-995(mouse fibrosarcoma), B-16 (mouse melanoma) and Meth-A (mousecarcinoma). Drug screening is carried out with a calorimetric cellviability assay in a 96-well plate.

[0132] Additionally, selected bioconjugates are radiolabeled with ¹⁴C or³H to assess the level of uptake by human tumor cells. As noted above,the prior art reports that some tumor and leukemia cells produce highlevels of B₁₂ binding proteins in the serum and sequester B₁₂ in highconcentrations of up to 50 fold.

[0133] Tumor Cell Line Testing Protocol:

[0134] The drug bioconjugates, synthesized under procedures describedherein or adaptions thereof, are diluted over 5 orders of magnitude(approximately 0.005 to 50 μg/mL). Four hours after seeding of the cellin the plate, the cells are treated with the appropriate drug dissolvedin isotonic buffered solution. In control experiments, withoutphotolysis triggered drug release, the drug is left on the cells forthree days, as in the normal basic cancer screen. In the wells fortriggered drug release, the laser output is focused on selected wellsfor varying times and with varying intensity. Alternatively, a matrix oflight-emitting diodes (LEDs) is used. The cells are incubated understandard mammalian tissue culture conditions under the proper CO₂balanced atmosphere. After three days, the cells are re-fed and thecalorimetric dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zoliumbromide (MTT) is added. The reduction of the MTT to purple formazanproduct is quantified in a 96 well plate reader. The concentration ofthe purple formazan dye is correlated with the number of viable cells.The reduction in cell survival at a given dose rate and photolysisexposure give a quantitative estimation of cell death and drug deliveryeffectiveness. Care is taken not to expose portions of the plate tophotolysis conditions through adventitious spillover of radiation. Thisis accomplished by using 96 well plates with an opaque mask (Fisher cat#07-200-565) for photolysis.

[0135] Uptake of Drug-B₁₂ and Drug-Co[SALEN] Bioconjugates:

[0136] The uptake of drug-B₁₂ and drug-Co[SALEN] bioconjugates bycultured tumor cells is monitored by radiolabeling the drug or cobalaminduring synthesis. ³H-Labeled 5-fluorouracil, methotrexate andchlorambucil are purchased from DuPont/NEN (New England Nuclear). Thesedrug bioconjugates, as well as ¹⁴C-labeled methylcob(III)alamin(synthesized from Cob(I)alamin and ¹⁴CH₃I) provide an indication ofreceptor-mediated uptake by the various tumor cell lines. In this study,the cells are exposed to the radiolabeled drug as described in thepreceding section, except no MTT is added at the end of the three-dayincubation period. Since all of the cell lines except the leukemia cellsgrow while attached to the bottom of the microtiter plate well, thegrowth medium is aspirated to remove the unincorporated radiolabeleddrug, followed by several washes with fresh medium. The labeled cellsare detached from the bottom of the wells and the radioactivityquantified by scintillation counting. Growth of non-attached leukemiacell takes place in round-bottom microtiter plates such thatcentrifugation sediments the cells and allows washing with fresh growthmedium before solubilizing the cells and quantifying the incorporatedradiolabeled drug by scintillation counting.

EXAMPLE 4 Synthesis of Co[SALEN]

[0137] Synthesis of N,N′-bis(salicylidene)ethylenediamine.

[0138] To a stirred solution of salicylaldehyde (12.21 g/10.62 mL) in70° C. ethanol (100 mL) was added ethylenediamine (3.01 g/3.33 mL). Ayellow crystalline material immediately formed, and the reaction mixturewas allowed to cool to room temperature with stirring. The solution wasfiltered, and the crystals were washed with cold ethanol. The ethanollayers were ombined and reduced to approximately 20 mL and allowed tostand at 0° C. overnight. The resulting crystals were collected byvacuum filtration and washed with water. The collected solids were driedin vacuo to obtain 13.15 g (98%) N,N′-bis(salicylidene)ethylenediamineas yellow platelets with a melting point of 126° C. (literature value(24) 127-128° c.). ¹H NMR (DMSO-d₆) δ 8.57 (s 2H, HC=N), 7.42 (d, 2Haromatic, J=7.3 Hz), 7.31 (t, 2H, aromatic, J=9.0 Hz), 6.88 (t, 4H,aromatic, J=8.3, 16.1 Hz), 3.89 (s, 4H, CH₂). ¹³C NMR (DMSO-d₆) δ 166.94(2C, CO⁻), 160.57 (2C, HC+N), 132.38 (2C, aromatic), 131.69 (4C,aromatic), 118.59 (2C, aromatic), 116.51 (2C, aromatic), 58.88 (2C,CH₂).

[0139] Synthesis of N,N′-bis(salicylidene)ethylenediaminecobalt(II)(Co[SALEN]).

[0140] To a hot (100° C.) deoxygenated solution of the above product(2.68 g) in dimethylformamide (25 mL) was added via cannula needle anaqueous solution (10 mL) of cobalt (II) acetate tetrahydrate (2.49 g).The red precipitate which formed was collected by vacuum filtration,washed with cold dimethylformamide, and dried in vacuo to obtain 2.6 g(80%) of N,N′-bis-(salicylidene)ethylenediaminocobalt (II) as redcrystals.

EXAMPLE 5 Synthesis of Modified Co[SALEN]

[0141] The diglycolate ether of Co[SALEN] is prepared as described inExample 4, using the glycolate ether of 2,5-dihydroxybenzaldehyde inplace of salicylaldehyde. An unsymmetrically substituted (glycolateether/amide) complex is prepared as described in Example 4 by usi g amixture of the glycolate ether of 2,5-dihydroxybenzaldehyde and5-aminosalicylaldehyde in place of salicylaldehyde.

EXAMPLE 6 Synthesis of Chlorambucil-Cobalamin Bioconjugates

[0142] Synthesis of1-bromo-2-[4-(4′-[bis-(2-chloroethyl)amino]phenyl)butyroxy]ethane.

[0143] Twenty-five mL of freshly distilled CH₂Cl₂, 0.343 gdicyclohexylcarbodiimide (1.66 mmol), 0.305 g 4-dimethylaminopyridine(2.5 mmol), and 0.263 g 4-dimethylamino-pyridinium chloride (1.66 mmol)were added to a flame-dried 50 mL round bottom flask equipped with astir bar, reflux condenser, and Ar inlet (Boden and Keck, 1985). Thesolution was purged with argon and brought to reflux. While refluxing, asolution of 0.304 g chlorambucil (1.0 mmol) and 0.125 g 2-bromoethanol(1.0 mmol) in 5 mL CH₂Cl₂ (purged under argon for 30 min.) wastransferred via cannula to the refluxing solution over a period of 30min. After addition was complete, the reaction mixture was stirred for 2h at room temperature. Precipitated dicyclohexylurea was removed byfiltration and the solution was concentrated by rotary evaporation. Theresulting residue was taken up in CH₂Cl₂, filtered, and purified byflash silica column chromatography. The desired product was eluted using1:9 ethyl acetate:hexanes (v/v) to give 0.374 g of a yellow oil in 91%yield (ester 2). ¹H NMR (CDCl₃, 300 MHz) d 7.06 (d, 2H, J=8.4 Hz), 6.60(d, 2H, J=8.7 Hz), 4.35 (t, 2H, J=6.15 Hz), 3.56-3.72 (m, 8H), 3.48 (t,2H, J=6.15 Hz), 2.56 (t, 2H, J=7.65 Hz), 2.35 (t, 2H, J=7.35 Hz), 1.91(quintet, 2H, J=7.58 Hz). ¹³C NMR (CDCl₃, 75 MHz ¹H decoupled) d 173.05,144.35, 130.37, 129.75 (2), 112.12 (2), 63.68, 53.55 (2), 40.60 (2),33.94, 33.41, 29.05, 26.72.

[0144] Synthesis of2-[4-(4′-[bis-(2-chloroethyl)amino]phenyl)butyroxy]ethylcob(III)alamin(3).

[0145] Two hundred mg of hydroxocob(III)alamin (0.15 mmol) was dissolvedin 10 mL water and purged with Ar while stirring (Brown and Peck, 1988).The exiting gas was conducted in sequence through: (1) a flaskcontaining 0.025 g NaBH₄ (0.66 mmol); (2) a flask containing 5 mL H₂O;and (3) a flask containing 0.226 g ester 2 (0.55 mmol) in 5 mL CH₃OH.After deaerating for 1 h, the water from flask (2) was transferred toflask (1) containing NaBH₄ via canrula and swirled to promotedissolution. This solution was transferred via cannula to the aqueouscobalamin solution. Reduction was allowed to proceed for 20 min, afterwhich the chlorambucil bromoethylester was added to the solution. Thereaction mixture was allowed to stir for an additional 5 min. and then0.2 mL acetone was added to destroy the excess borohydride. The solutionwas concentrated to approximately 2 mL by rotary evaporation and theresulting solution was applied to a 2.5×30 cm column of Amberlite XAD-2resin. The column was washed with 1 L H₂O to desalt and the coba aminwas eluted with 50% aqueous acetonitrile (v/v). The eluent was reducedto approximately 2 mL by rotary evaporation and the solution was appliedto a 1×40 cm column of SP-Sephadex C25 (Na⁺ form). Elution with waterremoved the major red band which was reduced to a minimal volume.Acetone was added until faint turbidity persisted after swirling. Thesolution was allowed to stand for 1 h at 0° C. and excess acetone wasadded to promote further crystallization. The crystals were collected byvacuum filtration and dried in vacuo. 3 was obtained as red crystals(122.5 mg) with a yield of 53%. MS (FAB+) calcd for C₆₈H₁₁₂N₁₄O₁₆CoPCl₂, 1541; found 1541.

[0146] 4-[4′-(bis-[2-chloroethyl]amino)phenyl]butyroylcob(III)alamin (4)was synthesized in a similar manner starting with the acid chloride ofchlorambucil and reating it with hydroxocob(III)alamin as above.

[0147] Synthesis of2-[4-(4′-[bis-(2-chloroethyl)amino]phenyl)butyroxy]ethyl-Co[SALEN] and4-[4′-(bis-[2-chloroethyl]amino)phenyl]butyroyl-Co[SALEN] are synthsizedin a similar manner using Co[SALEN] in place of hydroxocob(III)alamin.

[0148] Synthesis of2-[4-(4′-[bis-(2-chloroethyl)amino]phenyl)butyroxy]ethyl-(Co[SALEN]-folate)and 4-[4′-(bis-[2-chloroethyl]amino)phenyl]butyroyl-(Co[SALEN]-folate)are synthsized in a similar manner using Co[SALEN]-folate in place ofhydroxocob(III)alamin.

[0149] Synthesis of2-[4-(4′-[bis-(2-chloroethyl)amino]phenyl)butyroxy]ethyl-(greencorrinoid) and 4-[4′-(bis-[2-chloroethyl]amino)phenyl]butyroyl-(greencorrinoid) are synthsized in a similar manner using CH₃-Co(III)corrinoid (prepared by reacting methyliodide with the green corrinoid ofBrown et al. (1996) after it had been reduced with NaBH₄) in place ofhydroxocob(III)alamin.

EXAMPLE 7 Sonolysis of2-[4-(4′-[bis-(2-chloroethyl)amino]phenyl)butoxy]ethylcob(III)alamin (3)

[0150] The products released by exhaustive sonolysis, as described inExample 2, of compound 3 (prepared in Example 6) were isolated byreverse-phase HPLC (Rainin Microsorb C-18). Elution and separation ofthe sonolysis products were carried out with an increasing gradient ofacetonitrile (A) and 0.05 M phosphoric acid (B): initial condition 5% A:95% B, increased linearly for 10 min to 30% A and 70% B, maintained for2 min; followed by a linear increase to 70% A and 30% B over 10 min(Rinchik et al., 1993). The solvent was evaporated from each fractionand the products were extracted with CH₂Cl₂ and characterized by ¹H and¹³C NMR.

[0151] Sequential absorption spectra of aqueous 3 as a function ofanaerobic sonolysis at pH 7.4, 100 mM Hepes, saturating Ar, are shown inFIG. 4A. The absorbance at 374 and 520 nm decreases linearly as afunction of sonolysis time, and the absorbance at 316 and 420 nmincreases linearly, thereby indicating the reaction is zero order insubstrate concentration. The isosbestic points at 336, 390, 486 and 585nm are in agreement with those obtained through anaerobic photolysis ofCH₃-Cbl^(III). The absorption band at 374 nm is characteristic of a Co—Cbond, and its disappearance unambiguously indicates displacement of theaxial carbon ligand.

[0152]FIG. 4B shows the change in absorbance spectra following aerobicsonolysis of a 3 solution containing phosphate buffer. Different stableproducts are obtained under aerobic conditions. Because of the presenceof molecular oxygen, the released product was shown by NMR to be2-[4-(4′-[bis-(2-chloroethyl)amino]phenyl)butyroxy]ethan-1-al. ¹H NMR(CDCl₃, 300 MHz) 9.59 (s, 1H), 7.08 (d, 2H, J=2.9 Hz), 6.62 (d, 2H,J=2.9 Hz), 4.67 (s, 2H), 3.73-3.59 (m, 8H), 2.60 (t, 2H, J=7.5 Hz), 2.45(t, 2H, J=7.4 Hz), 1.95 (quintet, 2H, J=7.4); ¹³C NMR (CDCl₃, 75 MHz ¹Hdecoupled) 195.85, 173.09, 144.54, 130.43, 129.92 (2), 112.29 (2),68.73, 53.74 (2), 40.69 (2), 33.99, 33.13, 26.79. The decrease inabsorbance at 374 nm is linear with increasing sonolysis time indicatingthe reaction is zero order in substrate concentration.

[0153] The Co—C bond of CH₃-Cbl^(III) can be cleaved by sonolysis inaqueous solutions to produce the alkane and cob(II)alamin underanaerobic conditions or to produce the aldehyde andhydroxocob(III)alamin under aerobic conditions. Unlike photolysis andthermolysis that lead to direct Co—C bond cleavage, the predominantpathway for Co—C bond cleavage by sonolysis is through H. mediatedreduction of CH₃—Cbl^(III) to the labile 19 e⁻ CH₃—Cbl^(II−) speciesfollowed by dissociation to the closed-shell alkane and Cbl^(II), orthrough the reaction of HO. with CH₃—Cbl^(III) that leads to formationof hydroxocob(III)alamin as well as degradation of the corrin ring.

[0154] A parallel exists between the reactions of alkylcob(III)alarninunder the conditions of sonolysis and pulse radiolysis, (Blackburn etal., 1972) but without the need for expensive equipment. Although theviolent cavitation during sonolysis has sufficient energy to break theCo—C bond to produce the {R. .Cbl^(II)} radical pair by a dissociativepathway analogous to the photolysis of CH₃—Cbl^(III), (Endicott andNetzel, 1979; Chagovetz and Grissom, 1993; Natarajan and Grissom, 1996),alkylcob(III)alamins are not sufficiently volatile to be found in theextreme environment of the collapsing bubbles. Therefore, direct Co—Cbond cleavage by sonolysis is not possible in spite of the more than 80kcal/mol difference in bond-dissociation energy between Co—C and H—OH.

[0155] Anaerobic sonolysis of the Co—C bond is irreversible because aclosed-shell alkane is formed. Under aerobic conditions, the rate of H.reaction with O₂ is on the same order of magnitude as the reaction of H.with CH₃, thereby suggesting the closed-shell alkane, CH₄, should be oneof the end products of CH₃—Cbl^(III) sonolysis (Buxton et al., 1988;Baulch et al., 1992). In contrast, Co—C bond cleavage of CH₃—Cbl^(III),via anaerobic photolysis, is reversible from the {CH₃. .Cbl^(II)}radical pair.

[0156] In summary, the ability to form cob(II)alamin and theclosed-shell alkane without the use of chemical reductants and withoutthe use of electrochemical, photochemical, or pulse radiolysis equipmentmay be a useful method to promote activation of drug-cobalamin complexesin vivo.

EXAMPLE 8 Materials and Methods for in vitro Assays of BioconjugateActivity

[0157] Media Preparation

[0158] All media were purchased from Sigma and materials used tosupplement the media were purchased from Atlanta Biologicals. The HL-60cell culture was grown in an α-MEM media. The media was completed priorto inoculation by the addition of reagents to bring the final mediaconcentration to 7.5% w/v sodium bicarbonate, 10% fetal calf serum, 100μg/mL, penicillin and streptomycin, and 50 units/mL mystatin. McCoy'smedia, with sodium bicarbonate buffer, was used for HCT-116 cellculture. It was completed in the same manner with 8% newborn calf serumand 2% fetal calf serum. Completed media could be stored at 4° C. forseveral weeks. The culture medium was warmed to 37° C. beforeinoculation with cells. Stock Culture Preparation and Maintenance Stockcell cultures were started from ATCC cell lines. The original ATCC cellline was aliquoted in 10% DMSO and stored in liquid nitrogen. Stockcultures of 40 mL were grown and maintained in collaen-treated, sterile75 mL culture flasks purchased from Corning. The cultures were incubatedat 37° C. in a 5% CO₂ environment to maintain a pH of 7.1. Humiditywithin the incubator was maintained to prevent hypertonicity in themedia by placing an open pan of water in the bottom of the incubator.

[0159] The concentration of cells within the stock culture wascontrolled and cell concentrations were estimated in several ways. Themedia became more purple and subsequently orange as a result of cellmetabolism and metabolic byproducts that accumulated in the media. Thecells were also visually observed under microscope at 40× and 100×power. Normal HCT-116 cells appeared rounded, flat, and adhered stronglyto the walls of the culture flask. When the cells almost covered thebottom of the flask, the cell concentration was reduced. Normal HL-60cells appeared round, but were well differentiated and easily suspendedin the media. Changes in cell morphology were often indicative ofbacterial or fungal contamination. For the accurate determination ofcell concentrations, a Coulter Cell CounterTM was employed. Stockcultures were not allowed to grow to greater than 100,000 cells/mL. Bothof the cell lines were observed to have a doubling tirne of about 24hrs.

[0160] Assay Preparation

[0161] The assays were performed in collagen-treated, sterile 96-wellplates that were purchased from Corning. HL-60 cells were grown inround-bottomed wells (Coming catalog #25850) and HCT-116 cells weregrown in flat-bottomed wells (Corning catalog #25860). Cellconcentrations were measured by a Coulter Cell Counter™. Cells werediluted in bulk and loaded onto the plates with 200 μL in each well. Theassays were performed using approximately 25,000 HL-60 cells/well and40,000 HCT-116 cells/well.

[0162] Since HL-60 cells grow in suspension, the cell concentration wasmeasured and diluted directly. HCT-116 cells, however, adhere to thewalls of the flask and must be suspended by treatment with trypsin. A0.025 mg/mL trypsin solution was thawed immediately before use. The bulkculture medium was removed by aspiration and 2 mL of the cold trypsinsolution were added to the flask. The flask was agitated periodically topromote suspension of the cells. Care was taken to limit cell exposureto the trypsin solution to less than five minutes, since prolongedexposure will damage the cell membrane. When the cells were suspended,as observed by a microscope, 8 mL of media were added to inactivate thetrypsin. The cell concentration in the resulting suspension wasmeasured, the suspension diluted appropriately, and loaded onto the96-well plates. Once on the plates, the cells where allowed to adherefor 3 hrs before treatment with SFU or one of the derivatives.

[0163] Cell Growth and MTT Determination of Cell Viability

[0164] HL-60 cells were treated and placed in the incubator for 24 hrs.The plates were centrifuged and the supernatant was carefully aspiratedwithout disturbing the cell pellet. A 200 μL aliquot of media was addedimmediately. The cells were allowed to grow for 48 hrs. HCT-116 cellswere treated and allowed to grow undisturbed for 72 hrs. The culturemedium was removed by aspiration (after centrifugation in the case ofHL-60 cells). 100 mL of McCoy's media and 11 μL MTT dye were added. Thecells were incubated at 37° C. for 3 hrs. During this time, viable cellsreduce the MTT dye to purple formazan by the action of alcoholdehydrogenase. The cells were lysed by the addition of 100 μL of asolution 1.2M Hcl in 60% ethanol, thereby releasing the reduced dye intosolution. The absorbance at 405 nm was measured for each well using aBIO-RAD Microplate Reader (Model 450™/

EXAMPLE 9 In Vitro Activity of Chlorambucil-Cobalamin Bioconjugates

[0165] Thermal Stability of Bioconjugates in Media

[0166] It was noted that the chlorambucil bioconjugates 3 and 4(prepared in Example 6) have thermal lability. Thus, they are expectedto thermally decompose during the assay, perhaps before entering thecells or before release by photolysis. Thermal decomposition of bothbioconjugates was monitored by a UV-vis diode array spectrophotometer(HP8452) at 37° C. in water, cell-free media, and filtered media inwhich HCT-116 cells had been grown to a concentration of about 100,000cells/mL. Spectra were taken hourly for a total of 8 hrs. The presenceof intact bioconjugate was then determined by photolysis, 20 min, with ahigh-pressure mercury lamp. If photolysis had no effect on the spectrum,all of the bioconjugate was assumed to have decomposed.

[0167] In Vitro Assays of 3 and 4 Activity

[0168] Both bioconjugates were assayed against HCT-116, HL-60, B-16,Meth-A, and RD-995 cell lines. The assays were performed in the samemanner as described in Example 8 as modified herein. The B-16, RD-995and Meth-A cells lines are all Balb/c derived carcinoma lines which wereprovided by Dr. R. Daynes of the University of Utah. These cell lineswere grown in RPMI media which was completed with 5% fetal bovine serumand other media components as previously described. Both the B-16 andRD-995 cell lines were suspended in trypsin, as in the case of HCT-116cells. The Meth-A cells loosely adhere to the walls of the flask andgrow both attached to the flask and suspended in solution. These cellscould be completely suspended by successive washing of the flask wallwith media.

[0169] Assays were performed at cell concentrations of about 40,000cells per well, with the exception of the HL-60 assay which wasperformed at 25,000 cell per well. The HCT-116, B-16 and RD-995 cellswere assayed in flat bottomed, 96-well plates, while the HL-60 andMeth-A cells were assayed in round bottomed plates. Chlorambucil,unconjugated, was tested prior to the bioconjugates. The cells weretreated with the bioconjugates in both non-photolytic and photolyticconditions. The cells were incubated for three days (media was aspiratedand replaced after 24 h. in the case of the HL-60 cells) and theresulting viability measured by an MTT assay.

[0170] The MTT assay was somewhat altered for this experiment. Theculture medium was aspirated after 72 hrs. The Meth-A and HL-60 cellswere centrifuged prior to aspiration. Then, 100 μL of McCoy's media and11 μL of the MTT solution were added as before. At the end of 4 hours,the culture medium was aspirated a second time (following centrifugationin the case of HL-60 and Meth-A cells) and 100 μL of DMSO was added. TheDMSO lysed the cells releasing the MTT dye into solution. The absorbanceof each well at 450 nm was measured as before. The HCl/ethanol solutionpreviously used has a tendency to precipitate proteins from theresulting solution, which may give falsely increased absorbancemeasurements. The replacement of DMSO avoids this problem.

[0171] The concentration of chlorambucil and the bioconjugates werevaried from 0.04 μM to 400 μM within the assay. The cells were treatedwith the bioconjugates under dim, red lights to avoid photolysis.Non-photolytic conditions were maintained by wrapping the 96 well plateswith foil during the incubation periods. Photolysis was performed inblack plates with flat, clear bottomed wells (Costar catalog number:3603). These plates are sterile, collagen treated, and made of opticallyclear plastic. Growth of the cells in these plates did not show anydifferences to those grown in the normal clear plastic plates.Photolysis was achieved by an array of high intensity green LEDs(Hewlett Packard catalog number: 782-6124). The array was constructedfrom one of the black plates in which one LED was placed in each well.The LEDs could be turned on and off as vertical rows. In each assay, tworows of cells were left untreated as growth controls. One of these rowswas not photolyzed by the LEDs to demonstrate any unexpected effects ofphotolysis; irradiation did not demonstrate any effects on the untreatedcontrol cells. An empty plate was placed between the array and the assayplate to avoid heating the cells. Ten minutes of irradiation producedcomplete photolysis for the bioconjugate in cell-free media. The cellswere irradiated for 10 min during the assay. The time of irradiation,following treatment with drug, was determined by a timecourse assay. Theentire plate was treated with one of the bioconjugates at aconcentration equal to the IC₅₀ of chlorambucil in that cell line. Therows were irradiated separately one half hour after treatment and thenhourly.

[0172] Irradiation at 1 h after treatment demonstrated the greatestbioconjugate activity in all of the cell lines. Further assays wereperformed with irradiation one hour after treatment. In the case of theMeth-A cell line, the cells were transferred from the round-bottomedplate into the black plate for photolysis and then returned to the roundbottomed plate. The HL-60 cell line could not be tested under thesephotolytic conditions.

[0173] Results and Discussion

[0174] Both bioconjugates show significant thermal decomposition in bothwater and cell free media at 37° C. At the end of 8 hrs, photolysis hasno effect on the spectrum, indicating that all of the bioconjugate hasdecomposed. In the HCT-116 cell media both bioconjugates show fastinitial decomposition and are significantly stabilized at subsequenttime. Haptocorrin, a cobalamin binding protein is known to stabilizealkylcobalamins by several orders of magnitude. This protein is presentin the cell-free media from the added bovine serum. However, most ofthis protein in serum is saturated with cobalamin, so binding to thebioconjugates may be inhibited. It is known that several types of tumorcells secrete high amounts of cobalamin binding proteins, especiallyhaptocorrin. Thus, media in which cells have been growing has a higherconcentration of apo-haptocorrin. The initial fast decomposition of thebioconjugates represents the amount remaining after the saturation ofhaptocorrin in the media. The bound bioconjugates are significantlystabilized by haptocorrin and the haptocorrin complex associates anddissociates in a dynamic fashion in solution, especially in the presenceof significant concentrations of other cobalamins in the media. Thus,the bioconjugates are still susceptible to decomposition when not boundto haptocorrin. While haptocorrin does stabilize the bioconjugates byseveral orders of magnitude, slow decomposition is still seen during theassay. However, this assay does indicate that the bioconjugates arestabilized such that a significant amount is still intact during thetime of uptake and photolysis.

[0175] The data from the photolysis timecourse and activity assays aresummarized in FIGS. 5-9 for compound 3 in each of the cell lines tested.Similar results were seen for compound 4. In general, both bioconjugatesshowed similar uptake and photolysis behavior in the photolysistimecourse assay. The maximal photolytically induced toxicity is seenone hour after treatment with either of the bioconjugates. In all cases,photolysis of the bioconjugate demonstrated increased toxicity over thatof unconjugated chlorambucil. FIG. 5 shows that the chemotherapeuticdrug, chlorambucil, has an LD₅₀ of about 2 μM with respect to theHCT-116 cell line, whereas the bioconjugate shows no substantialtoxicity at concentrations approaching 100 μM. If cells treated with thebioconjugate are subjected to brief irradiation with red light 12 hoursafter dosing, the LD₅₀ decreases by a factor of 25 to 0.08 μM. If a10-fold excess of vitamin B₁₂ is added to saturate the cell surfacereceptors, the bioconjugate is not taken into the cells and photolysistriggers release of the active chlorambucil in the cell culture medium.The released chlorambucil now enters the cell by passive diffusion andan LD₅₀ of 2 μM is observed—in close agreement with the value forchlorambucil standard.

[0176]FIG. 6 shows that in cell line HL-60, the unconjugatedchlorambucil standard exhibits an LD₅₀ of 0.5 μM, but the bioconjugateis at least 2-fold better with an LD₅₀ of 0.2 μM. The cytotoxicity ofMC-121 against the leukemia cell line is still a dramatic result whencompared with the absence of toxicity when the cellular uptake of theconjugate is out-competed by the addition of 10 equivalents of vitaminB₁₂. Similar results were obtained with Meth-A cells. The HL-60 andMeth-A cells have a high turnover rate, and in the case of Meth-A dividemore rapidly than the other cell lines. These cells may, in fact,metabolize cobalamin at a faster rate than the other cell lines and thusrelease the chlorambucil in significant concentrations withoutphotolysis. In order for this to be practical, however, cobalaminmetabolism must occur before significant hydrolysis of chlorambucilmoiety. It is reported that HL-60 cells are able to convert vitamin B₁₂into the other cobalamin forms efficiently (more quickly than normallymphocytes) (Quadros and Jacobsen, 1995) and thus, would be able toefficiently release the conjugated chlorambucil. In the other celllines, however, the bioconjugates are essentially not toxic innon-photolytic conditions, which is a promising indication that thesebioconjugates may not be toxic in normal somatic cells or healthyhematopoetic cells. IC₅₀ values of the bioconjugates in bothnon-photolytic and photolytic conditions are summarized in Table 1.TABLE 1 IC₅₀ values (μM) Cell Line HCT-116 HL-60 B-16 Meth-A Rd-995Chlorambucil 20.8 5.8 1.4 1.8 1.1 Photolysis 3 1.7 — 0.6 0.2 0.2 4 1.1 —0.3 0.3 0.3 No Photolysis 3 — 3.2 — 210.1 — 4 — 8.9 — 84.2 —

[0177] It will be appreciated that the methods and compositions of theinstant invention can be incorporated in the form of a variety ofembodiments, only a few of which are disclosed herein. It will beapparent to the artisan that other embodiments exist and do not departfrom the spirit of the invention. Thus, the described embodiments areillustrative and should not be construed as restrictive.

LIST OF REFERENCES

[0178] Allen, R. H. and Majerus, P. W. (1972a). J. Biol. Chem.247:7695-7701.

[0179] Allen, R. H. and Majerus, P. W. (1972b). J. Biol. Chem.247:7709-7717.

[0180] Baulch, D. L., et al., (1992). J. Phys. Chem. Ref Data 21:411.

[0181] Bennett, C. F., et al. (1992). Mol. Pharmacol. 41:1023-1033.

[0182] Blackburn, R., et al. (1972). J. Chem. Soc. Faraday I, 1687.

[0183] Boden, E. F. and Keck, G. E. (1985). J. Org. Chem. 50:2394.

[0184] Brown, K. L. and Peck, S. (1988). Organomat. Syn. 4:304.

[0185] Brown et al. (1996). J. Inorg. Chem. 35:3442.

[0186] Bunnell, B. A., et al. (1992). Somatic Cell Mol. Genet.18:559-569.

[0187] Buxton, G. V., et al., (1988). J. Phys. Chem. Ref Data 17:513.

[0188] Carmel (1975). New Engl. J. Med. 292:282-284.

[0189] Castle, W. B. (1953). N. Engl. J. Med. 24:603-611.

[0190] Chagovetz, A. M. and Grissom, C. B. (1993). J. Am. Chem. Soc.115:12152.

[0191] Chang, E. H. and Miller, P. S. (1991). In Prospects for AntisenseNucleic Acid Therapy of Cancer and AIDS, ed. Wickstrom, E., Wiley-Liss,New York, pp. 115-124.

[0192] Chen and Chance (1993). Biochem. 32:1480-1487.

[0193] Cohen, J. S. and Hogan, M. E. (1994). Sci. Am. 76-82.

[0194] Cooney, M., et al. (1988). Science 241:456-459.

[0195] Endicott, J. F. and Netzel, T. L. (1979). J. Am. Chem. Soc.101:4000.

[0196] Fomari, F. A., et al. (1996). Biochem. Pharmacol. 51:931-940.

[0197] Fox, H. J. and Castle, W. B. (??). Am. J Med. Sci. 203:18-26.

[0198] Fritzer, M., et al. (1996). Biochem. Pharmacol. 51:489-493.

[0199] Gao, X. and Huang, L. (1991). Biochem. Biophys. Res. Commun.179:280-285.

[0200] Gasparro (1986). Biochem. Biophys. Res. Commun. 141:502-509.

[0201] Ginobbi, P., et al. (1997). Anticancer Res. 17:29-36.

[0202] Grissom, C. B. (1995). Chem. Rev. 95:3-25.

[0203] Hosada, J., et al. (1995). Biol. Pharm. Bull. 18:1234-1237.

[0204] Howard, W. A., et al. (1997). Bioconj. Chem. 8:498-502.

[0205] Hu, Y. -P., et al. (1996). Cancer Chemother. Pharmacol.37,556-560.

[0206] Johnson, D. A., et al. (1995). Anticancer Res. 15:1387-1394.

[0207] Kabanov, A. V., et al. (1995). Bioconjugate Chem. 6:639-643.

[0208] Krautler, B. and Stepanek, R. (1985). Angew. Chem. Int. Ed. Engl24:62.

[0209] Kumar et al. (1987). J. Biol. Chem. 262:7171-7179.

[0210] Le Doan, T., et al. (1987). Nucl. Acids Res. 15:7749-7760.

[0211] Ling, Y. -L., et al. 1996). Mol. Pharmacol. 49:832-841.

[0212] Letsinger, R. L. (1993). Nucl. Acids Symp. Ser. 29:1-2.

[0213] Longman, S. A., et al. (1995). Cancer Chemother. Pharmacol.36:91-101.

[0214] Lott et al., (1995). J. Am. Chem. Soc. 117:12194-12201.

[0215] Low, P. S. et al. (1995). U.S. Pat. No. 5,416,016.

[0216] Ma, D. D. F. and Wei A-Q. (1996). Leukemia Res. 20:925-930.

[0217] Madon, J. and Blum, H. E. (1996). Hepatology 24:474-481.

[0218] Mason, T. J. (1991). Practical Sonochemistry. Users Guide toApplications in Chemistry and Chemical Engineering Horwood, N.Y.

[0219] Merwin, J. R., et al. (1994). Bioconjugate Chem. 5:612-620.

[0220] Moser, H. E. and Dervan, P. B. (1987). Science 238:645-650.

[0221] Murray, G. J. and Neville, D. M. (1980). J. Biol. Chem.255:11942-11948.

[0222] Myers (1988). European Patent Application No. 86810614.7.

[0223] Natarajan, E. and Grissom, C. B. (1996). Photochem. Photobiol.64:286.

[0224] Nichols, J., et al. (1997). Eur. J. Cancer 33 Suppl. 1:S34-S36.

[0225] Oeltmann and Heath (1979). J. Biol. Chem. 254:1028.

[0226] Postel, E. H. (1992). Ann. N.Y Acad. Sci. 660:57-63.

[0227] Quadros, E. V. and Jacobsen, D. W. (1995). Biochim Biophys. Acta.1244:395-403.

[0228] Rando, R., et al. (1994). Nucl. Acids Res. 22:678-685.

[0229] Rinchik, E. M., et al. (1993). Bioessays 12:831-836.

[0230] Roth and Maddox (1983). J. Cell. Phys. 115:151.

[0231] Russell-Jones, G. J. et al. (1995). U.S. Pat. No. 5,428,023.

[0232] Sivam, G. P., et al. (1995). Cancer Res. 55:2352-2356.

[0233] Skoog, J. U. and Maher, L. J., III (1993). Nucl. Acids Res.21:2131-2138.

[0234] Stein, C. A. and Cohen, J. S. (1988). Cancer Res. 48:2659-2688.

[0235] Svensson, H. P., et al. (1995). Cancer Res. 55:2357-2365.

[0236] Szczylik, C., et al. (1991). Science 261:562-565.

[0237] Tanaka, T., et al. (1996). Biol. Pharm. Bull. 19:774-777.

[0238] Trubetskoy, V. S., et al. (1992). Biochim. Biophys. Acta1131:311-313.

[0239] Uhlmann, E. and Peyman, A. (1990). Chem. Rev. 90:543-584.

[0240] Vlassov, V. V., et al. (1994). Biochim. Biophys. Acta1197:95-108.

[0241] Wang, S., et al. (1995). Proc. Natl. Acad. Sci. USA 92:3318-3322.

[0242] Waxman, et al. (1972). Clin. Res. ??:572.

[0243] Weissler, A. (1962). Nature 193:1070.

[0244] Wickstrom, E., et al. (1992). Cancer Res. 52:6741-6745.

[0245] Wu, G. Y. (19887). J. Biol Chem. 262:4429-4432.

[0246] Yao, Z., et al. (1996). J. Viral Hepat. 3:19-22.

[0247] Zon, G. (1993). Methods Mol. Biol. 20:165-189.

What is claimed is:
 1. A method of administering a bioactive agent tocells of a targeted tissue site of a subject which comprisesadministering to said subject an effective amount of the bioactive agentas a bioconjugate, said bioconjugate comprises the bioactive agent andan organocobalt complex wherein the bioactive agent is covalentlyconjugated to the cobalt atom through a non-reactive atom in thebioactive agent molecule.
 2. The method of claim 1, wherein said cellsof said targeted tissue site have an affinity for the organocobaltcomplex portion of said bioconjugate.
 3. The method of claim 1, whereinsaid cells of said targeted tissue site have an affinity for thetargeting molecule of the organocobalt complex portion of saidbioconjugate.
 4. The method of claim 1, wherein said bioconjugate isadministered intravenously.
 5. The method of claim 1, wherein saidbioconjugate is administered parenterally.
 6. The method of claim 1,wherein said bioconjugate is administered orally.
 7. The method of claim1, wherein said bioconjugate is administered intramuscularly.
 8. Themethod of claim 1, wherein said bioconjugate is administeredintrathecally.
 9. The method of claim 1, wherein said bioconjugate isadministered as an aerosol.
 10. The method of claim 1, wherein saidtargeted tissue site is neoplastic tissue and said bioactive agent is ananticancer agent.
 11. The method of claim 10, wherein said neoplastictissue is tissue of a sarcoma.
 12. The method of claim 10, wherein saidneoplastic tissue is tissue of a carcinoma.
 13. The method of claim 10,wherein said neoplastic tissue is tissue of a leukemia.
 14. The methodof claim 1, wherein said targeted tissue site is tissue afflicted withpsoriasis and said bioactive agent is a cytotoxic agent oranti-metabolite.
 15. The method of claim 1, wherein said targeted tissuesite is tissue for the application of gene therapy and said bioactiveagent is an oligonucleotide or a polynucleotide.
 16. The method of claim15, wherein said oligonucleotide is antisense DNA or RNA.
 17. The methodof claim 1, wherein said targeted tissue site is tissue for theapplication of peptide therapy and said bioactive agent is a peptide orprotein.
 18. The method of claim 1, wherein a bolus of vitamin B₁₂ isadministered prior to administration of said bioconjugate.
 19. Themethod of claim 1, wherein nitrous oxide is administered first todeplete body stores of vitamin B₁₂.
 20. The method of claim 1, whereinsaid non-reactive atom is selected from the group consisting of a carbonatom, a nitrogen atom, an oxygen atom, a sulfur atom, a selenium atom ora silicon atom.
 21. The method of claim 1, wherein said non-reactiveatom is a carbon atom.
 22. The method of claim 1, wherein thenon-reactive carbon atom is a carbon atom from an alkyl, acyl or arylgroup that will not lead to rearrangement or destruction of thebioactive agent under conditions of ligand exchange duringreceptor-mediated endocytosis.
 23. The method of claim 1, wherein saidbioactive agent is covalently bound directly to the cobalt atom of theorganocobalt complex.
 24. The method of claim 1, wherein said bioactiveagent is covalently bound indirectly to the cobalt atom of theorganocobalt complex via a spacer.
 25. The method of claim 24, whereinsaid spacer is a self-destructing linker.
 26. The method of claim 1,wherein said bioactive agent is a diagnostic compound.
 27. The method ofclaim 1, wherein said bioactive agent is a drug.
 28. The method of claim27, wherein said bioactive agent is an anticancer agent.
 29. The methodof claim 1, wherein said bioactive agent is a peptide, peptide analogue,protein or protein analogue.
 30. The method of claim 1, wherein saidbioactive agent is a nucleic acid or a nucleic acid analogue.
 31. Themethod of claim 30, wherein said nucleic acid or nucleic acid analogueis a polynucleotide, oligonucleotide, antisense DNA or antisense RNA.32. The method of claim 1, wherein said organocobalt complex iscobalamin, a cobalamin derivative or a cobalamine analogue.
 33. Themethod of claim 1, wherein said organocobalt complex is a compoundhaving the following formula:

wherein the substitutents may be included or omitted to modulatephysical properties of the molecule, e.g., water solubility, stabilityor λ_(max)—the wavelength at which the complex absorbs.
 34. The methodof claim 33, wherein said targeting molecule is selected from the groupconsisting of glucose, galactose, mannose, mannose 6-phosphate,transferrin, cobalamin, asialoglycoprotein, α-2-macroglobulins, insulin,a peptide growth factor, folic acid or derivatives, biotin orderivatives, YEE(GalNAcAH)₃ or derivatives, albumin, texaphyrin,metallotexaphyrin, a vitamin, a coenzyme, an antibody, an antibodyfragment and a single-chain antibody variable region (scFv).
 35. Themethod of claim 1, wherein said organocobalt complex is selected fromthe group consisting of organo(pyridine)bis(dimethylglyoximato)cobalt, acorrinoid, derivatives thereof and analogues thereof.
 36. The method ofclaim 1, wherein said organocobalt complex comprises a multipleunsaturated heterocyclic ring system bonded to a cobalt atom through 4-5nitrogens and/or chalcogens which are part of said ring system.